University of Nigeria Research Publications
NNAMANI, Petra Obioma Author PG/M.PHARM/02/32723 Studies on Mucin Extracted from the Giant Snail Archachantina Marginata Title (Fam. Arionidea) Pharmaceutical Sciences
Faculty
Pharmaceutics Department
June, 2004
Date
Signature
S'FUDIES ON MUCIN EXTH ACTED FROM THE GIANT SNAIL ARCHACHA TINA M,4 IIGIM TA, FAM. ARIONIDAE
NNAMANI, PET1.U OBIOMA (PGIM. PHARM..102/32723)
DEPARTMENT OF PHARMACEUTICS, FACULTY OF PHARMACEUTICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA
JUNE, 2004 STUDIES ON MUCIN EXTR ' TEDFROM. THE GIANT SNAIL A RCHA CHA TINA MAH r;!lNATA, FAM. ARIONIDAE
NNAMANI, PETRA ORIOMA (PG/M. : IARM./02/3 2723)
A DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF THE MASTER OF PHARMACY (M. PEIARM.) DEGREE OF THE UNIVERSITY OF NIGERIA, NSUKKA.
SUPERVISOR: DR. M. U. ADIKWU
JUNE, 2004 iii
CER'I'I I (CATION
Nnanlani, Petra Obioma, a postgraduate :tr~dent in the Department of Pharrnaceutics and with the Reg. No. PGIM. PHARM.10213272 ' 1 ;satisfactorily completed the requirements for research work for the degree of Master of 1 harmacy in Pllarmaceutics. The work embodied in this dissertation is original and has not bep 1 submitkd in part or full for any other diploma or degree of this or any other University.
Dr. V.C. Okore (Head of Department) (Supervisor)
"\x!.kr, \~WQ&2 %L? Date:...... DEi 'ATION This work is dedicated to:
M'Y ' ..'H'I LD i 'EN ;
Eze-Nnamani, Cecil J. J. and Petra Ugochi~lyere~n,wl'hout whose companion this work would
have been cc !pleted much ~arlier.
M-.:!IUSBAND ;
Dr. f t!:l.il Eze Nnamani,
for his affection, inspira ' >n,understanding anti .;:.ern.
MY' L'4RENTS;
Mr. And Mrs. Gabriel Chukwuma Ukwucz . and Mr. And Mrs. Pius Ugwoke Nnamani for their
mcral support.
And,
MY SIELINGS ;
Engr. Bona, Sr. Ann, Mum. Doris, Jude, Felicia, Crescent, Vitalis, Lilian and Perpetua for their
understant ling and concern. v
ACKNOWI. ; BGEMENT
I am greatly indebted to my supervie ,r. ,. M. U. Adikwu for his expert advice, encouragement, assistance and constructive r riticisnl3 that made the successful completion of this work possible.
I am also greatly indebted to C\r. A. A. Attama for his invaluable assistance, encouragement, intelligent instructions, di~,%ctions and guidance throughout the period of the
work and as always. I could never have iniv I: it without you. I also thank Dr. C. 0. Esimone for his advice and encouragement. May God b ess you all.
. My sincere thanks go to Dr. F.0.C Nwodo for his kind assistance and advice. I wish also to express my appreciation to Drs. Iherlioha J.I. for his wonderful assistance in the work and friendly sharing of ideas always. I also wish '0 appreciate Dr. Charles Okoli, Pharms. Ogbonna
Okorie, Udeogaranya, Ezejiofor, Ofokansi, Mr. Uzuegbu Dave, Mrs. Ng Nwodo and Mrs. U. C.
Odo for their assistance and friendly sharing of ideas during the period of the work and as always. I also immensely thank Prof. P. A. Akah and Dr. G. C. Onunkwo for their support and co-operation. My sincere gratitude also goes to Mr. Awa Uka, Mr. Ogboso and other staff of the
Department of Pharmaceutics for their kind assistance in many areas of the work. I must as a matter of necessity acknowledge the warmth of some friends and colleagues Pharms. Nzekwe,
Bzea, Ike-Uno, Onugu, Dieke, Nwozuzu, Izuegbu and Nnaeto.
Finally, I thank God for my life, my family, well-wishers and more especially for the successful completion of this work. However, it is difficult to acknowledge everyone who lent a helping hand in one way or the other to see this research completed successfully. I only pray
God to bless and reward all of them abundantly.
Nnamani, P. 0. (Nee Ukwueze) TABLE OF ('ONTENTS
Title Page ... ii ... Certification ... 111 Dedication ... iv Acknowledgement v Abstract xii Table of contents vi CHAPTER ONE 1.0 Introduction 1 I . I Bioadhesion 1 1.1.1 Mechanism of Bioadhesion ... 1 1.1.2 Forces of adhesion ...... 3 1 . 1.3 Factors affecting bioadhesion ... 4
1.1.3.1 Mucin - mucin cohesive forces 4 1.1.3.2 Molecular weight of adhesive material 4 1.1.3.3 Coat weight ...... 5 1.1 3.4 Hydrogen ion concentration ...... 5 1.1.3.5 Hydration of adhesive material ... 5 1.1.3. G Presence of electrostatic charges ... G 1.1.4 The bioadhesive mucus ...... 6 1 . 1.5 Adhesive polymers ...... 7 1 I 0 Carboxyvinyl polymer (carbopolTM)... 9 I . I .0. I Carbomer (Carbopol Ultrez- I o'~)... 10 1.1.7 Sodium carboxymethylcellulose ... 11
1.1.8 Tragacanth ...... , 12
1.1.9 Gelatin ...... a 12 1.1.10 Methods of measuring bioadhesive strength of polymer 12 vii 1.1.1 1 Applications of bioadhesion ...... 14
1.2 Dissolution ...... a ...... 15 1.2.1 Methods of studying dissolution ...... 16 1.2.1.1 Modified USP disintegration apparat~ls ...... 16 1.2.1.2 The rotating basket assembly ...... 16 1.2.1.3 The paddle mixer ...... 17
1.2.1.4 Rotary impellers ...... a...... 18 1.2.2 Factors affecting dissolution rates of dnrgs in t.!e gastrointestinal tract ... 19 1.2.2.1 Physiological condition ...... 19 1.2.2.2 Physicochemical properties ...... 20 1.3 Gastrointestinal motility ...... 23 1.4 Action on the peripheral nervous system ...... 24
1.5 Medicament ...... 25 1.5.1 Chlorpropamide ...... 26 1.6 Viscosity. rheology and the flow of fluids ...... 27 1.6.1 Newtonian fluids ...... 28 1.6.1.1 Dynamic viscosity ...... 28 1.6.2 Non-Newtonian fluids ...... 31 1.6.2.1 Types of non-Newtonian behaviour ...... 31 1.6.3 Methods used in determining the flow properties of simple fluid ...... 33 1 h.4 Determination of the flow properties of non-Newtonian fluids ...... 33 1.7 Gel permeation with Sephadex ...... 34 1.7.1 Crosslinked dextran (Sephadex) properties ...... 35 1.7.1.1 Physical properties ...... 35 1.7.1.2 Chen~icalstability of Scphadcx ...... 36 1.7.2 Applications of Sephadex ...... 36 1.7.2.1 Analysis of multi-component samples ...... 36 1.7.2.2 Determination of molecular weights ...... 37 1.7.2.3 Zone electrophoresis ...... 37 1.8 Mucins ...... 38 1.8.1 The biological mucus membrane ...... 39 viii
1.8.2 Cl~aracterizationof much ...... 1.8.3 Interaction of mucin with biological con~pounds ...... 1.8.4 A new glycosaminoglycan from the giant African snail Achafinafilica 1.9 Snails ...... 1.9.1 The giant land snails of West Africa ...... 1.9.2 The genus Ackatincr ...... 1.9.3 Achatina (Achatina) achatina (Linnk) ...... 1.9.4 Common species of the genus Archachatina ... 1.9.4.1 Archachatina (calachatina) inarginata (Swainson) 1.9.4.2 Archachatina (Calachatina) dcgreri Bequaert ... 1.9.4.3 Archachatina (calachatina) ventricosa (Gould) 1.10 Preservation of snails ...... 1.1 1 'I'he Giant African snail: Achntinnficlica Fen~ssae 1.12 Physical requirement and production ...... 1.13 'The control of Achntina fulica ...... 1.13.1 Chemical control ...... 1.13.2 Collection and destruction ...... 1.13.3 Other control and protective measures ... 1.13.4 Biological control ...... 1.14 In search of protein: Snailery ... 1.15 Analgesic potentials of snail ... 1.16 Objectives of the study ...
CHAPTER TWO 2.0 MATERIALS AND METHODS 2.1 Materials ...... 2.1.1 Snails ...... 2.1.2 Animals ...... 2.2 Methods ...... 2.2.1 Extraction of snail mucin ... 2.2.2 Pliysicochemical tests ...... , ...... 2.2.2.1 Test for sugars ...... 2.2.2.2 Test for carbol~ydrates(reduction test) ...... 2.2.2.3 Test for proteins (amines; oxidation test) ...... 2.2.2.4Test for fixed oils ...... 2.2.2.5Solubility profile of the snail inucin ...... 2.2.3 Preparation of snail rnucin dispersion ...... 2.2.4 Rheological study on aqueous snail mwin dispersion ......
2.2.5 Type of flow ...... a,...... 2.2.6 Effect of concentration of the snail much ...... 2.2.7 Effect of temperature ...... 2.2.8 Effect of electrolytes ......
2.2.9 Effect of polymers ...... , ...... 2.2.10 Effect of aging ...... 2.2.1 1 Molecular weight determination by gel permeation chromatography on Sephadex G-200 ...... 2.2.12 Determination of snail mucin isoelectric point ...... 2.2.13 Test for allergic properties ...... 2.2.13.1 Preparation of stock solution ...... 2.2.13.2Guinea pig wheal test ...... 2.2.14Toxicological studies ...... 2.2.14.1 LDSodeterminatioil ...... 2.2.14.2Chronic toxicity determination ...... 2.2.15 'I'ensiometric determination of bioadhesive strength of snail mucin ... 2.2.15.1 Preparation of snail mucin stock ...... 2.2.15.2The bioadhesive strength ...... 2.2.16 Evaluation of the bioadhesive strength of the snail mucin using coated glass beads ...... 2.2.16.1 Preparation of simulated gastric fluid (SGF) without pepsin ... 2.2.16.2 Preparation of simulated intestinal fluid (SIF) without pancreatin ... 2.2.16.3 Coating of glass beads ...... 2.2.1 6.4 The bioadhesion test: Use of coated glass beads 2.2.17 Bioadhesion of snail mucin granules ...... 2.2.17.1 Preparation of granules ...... 2.2.17.2 Bioadhesion test on granules ...... 2.2.18 Determination of the granule micromeritics ... 2.2.1 8.1 Flow rate and angle of repose ...... 2.2.18.2 null; and tapped densities ...... 2.2.19 Gastrointestinal motility test in mice ...... 2.2.20 Determination of hmax of absorption ...... 2.2.21 Calibration curve of chlorpropamide in SIF (Beer's plot) 2.2.22 Absolute drug content ...... 2.2.23 Release studies ......
CHAPTER THREE RESULTS AND DISCUSSION ...... Physicochemical properties of snail much ...... Rheological test on aqueous dispersion of snail mucin
Type of flow ...... a. ... Effect of concentration of the much ...... Effect of temperature ...... Effect of electrolyte ...... Effect of polymer ......
Effect of aging ...... a...... Molecular weight determination ...... lsoelectric point determination of the snail mucin Local anaesthetic property ...... Toxicological studies ...... Acute toxicity test ...... Chronic toxicity test ......
3.6.2.1 Haematology ...... , 3.6.2.2 Histopathology ...... 3.7 Tensionietric determination of bioadhesive strength ...... 3.8 Evaluation of bioadhesive strength using coated glass beads ... 3.9 Bioadhesive test on grmrlcs ...... 3.10 Granule micromeritics ...... 3.1 1 Effect on small intestinal motility ... 3.12 Absolute drug content of granules ... 3.13 Release studies ......
CHAPTER FOZJR SUMMARY AND CONCLUSION ... REFERENCES ...... APPENDICES ...... xii
A DS'I'RACT
Snails have been eaten since prehistoric times. They have many roles in folk medicine.
Snails are 11ighly proteinous and rich in iron. Snail shells when crushed can be used for the reinforcement of cement for building. This research work was carried out to determine the possibility of utilizing snail mucin in drug forniulation. Snail mucin was extracted from the giant
African snail, Archnchntinn mnrginntn, Fani. Arionidne, by selective washing with distilled watcr and lyophilization of the aqueous extract. The lyophilized snail mucin was sub.iected to some physicochemical tests such as test for carbohydrates, sugars, proteins, fats and solubility.
Rheological studies were carried o~ton the snail nlucin, and the effects of concentration, electrolytes, polymers, aging and temperature on the viscosity of the snail much dispersion
detennined. Tlw molecular weight of the snail niucin was determined by gel permeation chromatography on Sephadex G-200, and the isoelectric point was also determined. The allergic
and toxicological properties (acute and chronic) and the effect of snail inucin on intestinal
motility were investigated. The bioadhesive properties of the snail mucin dispersion were
evaluated using a tensiometer and coated glass beads. Granules containing chlorpropamide
admixtures of snail mucin and Carbopol Ultrez-I0 were fortnulated and evaluated and their
bioadhesiveness also determined. The release properties of chlorpropamide from the granules
were studied in sitnulated intestinal fluid.
The results of the studies indicate that snail mucin contains carbohydrates, sugars,
proteins and fats. It is insoluble in organic solvents and aqueous acids and alkalis, but forms
dispersion in water. The snail mucin dispersion possesses pseudoplastic flow behaviour. It is
affected by increase in temperature at~daging. The nlolecular weight and the isoelectric point ... Xlll were fbund to be 4,281 Daltons and 3.4 respectively. 'The snail mucin was found to be non-toxic
as there was no serious effect on the tested ~j:ans and haenlatological parameters monitored.
711c snail mucin dispersions of 16 and 20 O/o W/Vhad the highest bioadhesive strengths of 100 % each, and the strength was found lo be maximal when simulated gastric fluid was used as the washing fluid. 'l'he Forinulated granules also had high bioadhesive strengths and possessed good
conlpressibility properties. The release of chloi-propamide from the granules varied depending on
the proportion of the snail muci~l/Carbopol Ultrez-10 admixtures. Analysis of the release
mechanisn~indicated that diffusion was the predominant mechanism of release.
From the results of the study, snail nlucin may prove to be a potential pharmaceutical
excipient either alone or in combination with other polymers. CIIAWER ONE
1N'I'RODUCTION
1.1 BIOADHESION
Rioadhesion is a state in which two malerials at least, one of which is of a biological nature, are held together for extended period of time by iiiterfacial forces [I]. For drug delivery purposes, the tcmi bioadhesion implies attachment of a drug carrier system to a specific biological location, which can be the epithelial tissue or the mucous coat on a tissue surface [2], thus increasing drug absorption and overall bioavailability [3-51. There are several types of controlled release bioadhesive dosage forms in use, some of which include: oral, buccal and nasal lioadliesive controlled release devices [GI. Rioadhesive dosage forms are targeted at particular sites such as nasal, buccal, gastrointestinal tract (Crrr), cervical, vaginal and dermal regions to reduce toxic side effects and increase the therapeutic efficacy of drugs [7].
1.1.1 Mechanism of bioadhesion
Polymer -- mucus bioadhesion have been extensively explained by the following theories: - the electronic, adsorption, wetting, diffusion and fracture theories [8]. However, due to the diversity in niechanisni involved in this interaction, no single theory has explained fully all the pl~enorr~enaobserved in bioadhesion.
The electroilic theory of Derjaguim and Smilga [9] states that upon contact of two niaterials with different electronic structures, there is electron transfer, which leads to the formation of a double layer of electrical charges at the adhesive interface. The attractive forces across the double layer are responsible for adhesion. The adsorption theory states that the materials adhere to each other because of van der
Waals, hydrogen bonding and other similar forces [8, 10,l I].
The wetting theory is largely applicable to liquid adhesives. This is related to interfacial tensions between the liquid bioadhesive materials to displace other materials that have been present in the stomach [12]. Such bioadhesive materials on contact with the surface must form a zero or near zero contact angles, a relatively low viscosity and should make an intimate contact that excludes air entrapment [13]. The surface tension, of the bioadhesive material and tissue will influence the extent of wetting. For a bioadhesive material to displace the gastric content and adhere spontaneously on the tissue, the spreading coefficient must be positive [ 131.
The interpenetration of macromolecular chains at the polymer-polymer interface at a temperature higher than the glass-transition temperature is the basis of the diffusion theory of polymer atlhesion proposed by Voyutski [13]. The molecular bridges which result from polymer self-diffusion account for the adhesive strength [12].
The fracture theory of adhesion can be applied to polymers in contact with soft tissue and relates the difficulty of separation of two surfaces after adhesion to the adhesive bond strength [13]. The characteristic feature of the mucus itself plays a very important role in the adhesion of polymers onto the mucus membranes. Lehr et al. [14] noted that drug delivery through nlucosa of the GIT is rate limited by mucus-turnover rather than by tightness of polymer adherence to the mucus lining. The mucus lining exists in different forms along the
GIT, respiratory tract and reproductive system. It also varies at different site within the same tract or system. The site at which the attachment is strongest is regarded as the optimal site for bioadhesion. This site is also expected to have : v mucus turnover rate and low sensitivity to stimuli that might enhance mucus secretion [15]. Irl :rease in the thickness of mucus decreases bioadhesion. IIence, the adhesion of polymer lo the arc as with thinner mucus layer e.g. caecum are always stronger than those with thick mucll-. layers stomach. The polymer-mucus layer interaction in the caecum for example is reater than the polymer-polymer molecule
interaction. In other words, the force of adhesion b:tween a polyrr~ and thinner mucus Ievel is greater than the force of cohesion of molec~~les.'I I\e pH of both the mucus and the polymer
affect the adhesion of the polymer.
1.1.2 Forces of adhesion
Mucus has adherent quality that makes it adhere .ightly to the food or other particles
[IG]. The process of mucoadhesion has been proposed t.) begin with the establishment of an
intinlate contact between the mucoadhesive polymer and the mucus gel [17]. The importance
of surface energy thermodynamics otherwise known as the work required in increasing the
surface area by I m2 in mucoadhesion has bcl recugnised as an important factor in
establishing an intimate contact [18]. This explains the physical or mechanical bond that
results on deposition and i~~clusionof an adhesive material in the crevices of the tissues.
Subsequently, the penetration of mucoadhesive polymer into the mucus gel network followed
by fonnation of secondary chemical bonds between the mucoadhesive materials is influenced
by several factors, which include the ionic charge of the polymer and the strength of the
hydrogen bonding between the polymers [I 9,1201.
'The hydrogen bond, which is a secondary chemical bond, is formed by compounds
containing hydrophilic functional groups such as hydroxyl, carboxyl, sulphate, amino groups (- OH, COOE-1, -SO4, -NH2)respectively [8]. Van der Waal interactions are classified based on the Debye forces due to permanent dipole-induced dipole interactions, Keesom forces due to permanent dipole-permanent dipole interaction and London forces due to induced dipole- induced dipole interactions.
1.1.3 Factors affecting bioadhesion
Several factors have been identified to influence the interfacial stability in bioadhesion.
These factors include:
1.1 J.1 Mucin-mocin cohesive forces
Mucus gel is a thick secretion composed mainly of water, electrolyte and a mixture of several glycoproteins, which themselves are composed of large polysaccharides [lb]. This mucus gel is held together by either primary disulfide bonds or secondary bonds (electrostatic and hydrophobic interactions) [8]. These cohesive mucin-mucin forces are the rate-limiting step in bioadhesion of several polymers [I 71. Therefore, the bioadhesive bond depends on the strength of the nlechanical bonds within the mucus. Heqce, the higher the mucin-mucin cohesive forces, the lower the bioadhesive bond force.
1.1.3.2 Molecular weight of adhesive material
Investigation on the effect of molecular weight of four viscosity grades of sodium carboxymethylcellulose (SCMC) by Kellaway el al. [18] showed that molecular weight affects bioadhesion and that optimum is 8, 600 Daltons. Polymers with molecular weight greater than
100,000 Daltons exhibit maximum adhesion [16]. 1.1.3.3 Coat weight
Increased coat thickness expressed as coat weight by Kellaway et al. [18] was found to cause considerable increase in adhesive forces. It is believed that inci-ease in coat weight will prevent over hydration of the polymer. Over hydration occurs in the presence of excessive quantity of water such that in this state the polymer losses all its adhesive properties.
1.1.3.4 Hydrogen-ion concentration
The concept of pH has been introduced as a more convenient measure of hydrogen ion concentration in a given material. A low pH has been observed by Kellaway et al. [I81 to favour adhesion. The adhesive characteristic of anionic polymers such as sodium laurylsulphate depends on pH and this decrease when the pH is greater than the pKa.
pH has several important applications in pharmaceutical practice. For example, it determines the solubility of a given material, the stability, viscosity; hence, it affects the adhesive properties of some material.
1.1.3.5 Hydration of adhesive material
Mucus is composed of among other things, water [14] and in the presence of mucus, adhesive material absorbs water from the mucus. Equilibrium should occur between the adhesive material and the mucus for bioadhesion to take place otherwise slippery mucilage leads to loss of adhesion. Tacky film gives maximum adhesion than hydration to form slippery mucilage [21].
Provided the time allowed for interaction between the mucus and the mucoadhesive material does not exceed certain limit, the contact time is directly proportional to the strength of the force of adhesion. Over hydration during this period should be prevented as noted by Chen and Cyr [2 I] to avoid loss of adhesion.
1.1 -3.6 Presence of electrostatic charges
Co~lsideringthe fact that mucus is ~~eg,tiivelycharged, any mucoadhesive material with a net positive charge e.g. gelatin will produce a relatively high degree of bioadhesion of longer duration, while a mucoadhesive polymer such as carboxymethylcellulose (CMC) with a net negative charge will produce a relatively low degree and duration of bioadhesion [21].
Different parts of the body secret mucus with different degree negativity.
Park and Robinson [22] studied the binding of various polymers to the mucin and epithelial cell surface and noted the importance of ionizable group. They found that polymers with ionizable groups were generally most adhesive.
1.1.4 The bioadhesive mucus
Mucus is a thick secretion from the mucus gland located on the surface of epithelium in most parts of the GIT [16]. It is composed mainly of water, electrolytes, and mixtures of several glycoproteins, which themselves are composed of large polysaccharides bound with much smaller quantities of protein. Mucus has been described as a gel or high viscous solution, which adheres to the luminar surface of the GIT by Niibuchi et a!. [23]. Mucus is also a highly viscous product, which forms a coat over the lining of hollow organs in contact with the external media [8]. Mucus is slightly different in different parts of the GIT, but everywhere, it has several important characteristics that make it both an excellent lubricant and a protectant for the wall of the gut. This slight difference is as a result of variation in the composition of the mucus from different origins. The mucus secretion with its main component as the glycoprotein fiaction is responsible for the gel-like characteristic [8]. Each glycoprotein consists of approximately 20 ?4 protein and 80 % sugar with an average molecular weight of about 2.14 x 10%altons 1241.
The mucus gel is held together by either primary (intra-chain disulfide bond) or
secondary (electrostatic and hydrophobic interaction). These glycoprotein molecules aggregate or associate with each other by means of non-covalent interaction forming the gel matrix
responsible for the rheologicat properties of the mucus [8]. This rheological property makes it
possible for bioadhesive polymers to i:.!eract well with the nucus leading to effective
bioadhesion.
1.I .5 Adhesive polymers
Polymers are substances of high molecular weight, consisting of repeating monomer
units [25]. The chemical and physical properties depend among other things, on their sizes,
symmetry and arrangement of the monomer units. The monomer units may be branched as in
amyiopectin or linear as in amylose while some: substances such as starch consist of both linear
and branched monomer units. Polymers exist in different forms and in pharmacy, polymer of
natural, semi-synthetic and synthetic sources are employed. These can be classified either as
water-soluble or water-insoluble polymers. The fonner has an ability to increase the viscosity
of solvents at low concentration to swell or change shape in solution and to absorb unto
surfaces [26]. It is the combination of slow solution rate and the formation of viscous surface
laycr that make hydropllilic polymers usef~ilin controlling the release rate of soluble drugs.
Tlic latter has a low rate of solution hence are used as membrane for dialysis or filtration, to form thin film coating materials, as packagi materials or to form matrices for enveloping drugs to control their release propc .i.7]. Examples include polyethylene, polyvinyl chloride, methylacrylate-methacrylate co-po er: ~dethyl cellulose.
'Three major categories have been util~,: r! ;uc:c:cssfully as bioadhesives [a].
1. Carboxyl-containing polymers
2. Hydroxyl-containing polymers
3. Polyniers with charged species polymers
Park er nl. 1223 found that cationic polymers exhibi1l.d low values of binding potential while anionic polymers exhibited high values. Also, among the ; qionic polymers, it was found
that those with carboxyl groups showed the highest binding pot. lia' Several neutral but
hydroxyl-containing polymers also exhibited high binding potcnlials. Introduction of
amphiphilicity property to a low nlolecular mass polymer by incorporating a hydrophilic
moiety into the molecule has been reported to improve the bioadhesive properties [26]. This
was observed to be due to increase in surface activity and seconda~ybond forming capacity due
to the increase in molecular chain length. Numerous polymers have been investigated for
bioadhesive properties and they include polysaccharides - acacia gum (gum arabic), tragacanth,
alginates, and water-soluble celluloses -- methylcellulose, hydroxymethylcellulose, sodium
carboxymethylcellulose (SCMC), hydrated silicates and magnesium aluminium silicate
(Veegum) [28]. 9 1.1.6 Carboxyvinyl polymer /Carbomelq(Carbopolm)
Carboxyvinyl polymer (C'arbopolTM)is a high molecular weight polymer up to about
3,000,000 Daltons. It is a synthetic product with a cross-linked polymer of acrylic acid co- polymerized with approximately 0.75-2 % of ally1 sucrose containing a high proportion of carboxyl groups f291. Its aqueous solution is acidic. When neutralized, the solution becomes very viscous with maximum viscosity between pH G and 11. Carbomers are anionic and electrolytes reduce their viscosity. Thus a high concentration of the polymer has to be employed in vehicles where ionizable drugs are present. They loose their viscosity on exposure to sunlight and this can be prevented or minimized by the addition of antioxidants.
Carbomers are used as suspending agents in pharmaceutical preparation and as binders in tablets. They are used in the formulation of prolonged acting tablets or sustained release tablet. Carbomers exist in various types such as 940, 941 and 943. There is also Carbopol ultrez-10. The first two are white flutty, acidic, hygroscopic powder with slight characteristic odour [29]. The difference in the molecular weight of Carbomers accounts for these forms.
For instance, Carbon~er941 has a molecular weight of 4 x 10~altonswhile that of Carbomer
940 is 1 x 10"altons [29]. They are soluble in water, and solutions of sodium hydroxide, potassiunl hydroxide, borax, amino acids, tetraethylalcohol, lauryl and stearyl amines
Some chemicals like benzoic acid, sodium benzoate and benzalkonium chloride decrease the viscosity of Carbomer dispersion. Carbomer 940 and 941 are incompatible with plienol, cationic polymers, strong acids and maximum concentration of electrolytes. It is discoloured by resorcinol and microorganisms grow well in an unpreserved aqueous dispersion. 10
1.1.6.1 Carbomer 10 (~arbo~ol@ultrezTM 10)
Carbopol ultrez 10 polymer is a new "universal" polymer in the Carbopol family. It is exceptionally easy-to-disperse polymer thnt offers a wide range of performailce properties and can be used in a variety of personal care qpplicatinn. While various rheological additives are selected for their unique properties in a par tr application, such as a gel, lotion or a cream,
Carbop01 ultrez 10 can be used in all three. Because of its unique dispersion properties,
carbopol ultrez 10 allows greater versatility ' 11 formulating and processing 1301. Carbopol ultrez 10 polymer provides the formulation fli {ibility of a multi-use rheological additive. The superior dispersing properties of Carbopol ~~!i,-ez10 polymer makes it a rheological additive, which is much easier to process and call also lead to time savings in production. Since
Carbopol ultrez I 0 polymer "wets" extremely qui :kly, it requires less time and effort to achieve a lump-free dispession. It is even possible to cc'qpletely wet Carbopol Ultrez-10 polymer without any mixing. When using Carbopol Ultrez-I(: polymers in an aqueous or gel product, the polymer can be added directly to the water [30].
Because Carbopol Ultrez-10 has been designed to !?ive highly efficient thickening in emulsions (similar to Carbopol 934), its structure can make it wore vulnerable to ions, such as the salts found in surfactants and in vegetable extracts. Therefore, when formulating products with high levels of ions, such as shampoos, bath or cleansing gels or aloe Vera gels, it is highly
I-ccommended to use Carbopol ETD 2020 resin, which has excellent performance properties in the presence of ions [30].
Carbopol Ultrez-I 0 polymer systems are viscoelastic with a pseud litic rheology, as is the case for most carbopol resins. The three-dimensional network structure of the closely packed, swollen nlicrogels provides a significant resistance to flow, or viscosity. The polymer ~nicrogelsmaintain their closely packed structure UU~:r low shear stresses. Once an applied stress excceds the critical yield stress, the polym I micrc gels begin moving past each other and the bulk gel begins to flow. The combinatic of a lov number of entanglements between particles and the exhibited yield value account.: for the short "buttery" flow of Carbopol Ultrez-
I0 polymer, which may be considered a hybr ,t between a Carboy.,' "40-type (more rigid) and a
( 'arbopol934-type (softer, more deformable) -1n [30].
Although Carbopol IJltrez-10 polymer 1s a new addition to the Carbopol family, it has been evaluated in a wide range of very differen. applications including gels, lotions and creams.
1.1.7 Sodium carboxymethylcellulose (SCP "J)
SCMC is a semi-synthetic polymet. It i: sometimes referred to as sodium cellulose glycolate [28]. It has n~olecularweight of !"),000 - 700,000 Daltons. It is produced when sodium monochloroacetate reacts with alkalinized -.ellulose giving sodium chloride and
glycolate as bye products 1281. SCMC is a white I( faintly yellow coloured, odourless,
hygroscopic powder or granular material having a faint papc .-like taste. It is soluble in water at
all temperature giving a clear solution. It is practically insotlible in most organic solvents.
Aqueous solution exhibits pseudoplastic flow behaviour. Steri1izi:tion such as irradiation leads
to decrease in viscosity of the powder or granules and also solution pH of between 5 and 10
[25] affects its viscosity and stability. It is incompatible with strongly a-;J;r: solutions and with
soluble salts of iron and some other metal such as aluminium, merc ild zinc [28]. It is
commonly used as a suspension stabilizer, coating agent, viscosity enhctlltcing agent and tablet
billder ;md disintegrant. It is commercially available in three viscosity grades namely high, 12 mcdium and low with viscosity being directly 1 >ncl';nnal to the degree of polymerization [=I
1.1.8 Tragacanth
It is produced from dried gummy exudate o (J,abilarderc) and other Asiatic species of Astrng, /!a. Tragaca~lihis a white to weak yellow translucent, odourless substance with an insipid mucilaginous task It swells in water to produce nearly uniform opalescent mucilage. It i insoluble in alcohl 'Tragacanth is used as an cniulsifier, viscosity enhancer and suspending aj;ent [28]. Also, it is used as demulcent, adhesive for denatures and a base for medicament ; :I for lubricants and surgical instruments. 1.1.9 Gelatin Gelatin is a purified protein obtained either by partial acid hydrolysis (type A) or partial alkalilie hydrolysis (type R) of animal collagen; it mny also be a mixture of the two types. Gelatin is a faintly yellow to light yellowish-brown tasteless solid, usually occurring as translucent sheets, shreds, granules or powder. It is pi actically insoluble in common organic solvents; it swells in cold water and on heating it gives a colloidal solution, which on cooling forms a more or less firm gel. The isoelectric point of ty. i gelatin is between pH 6.3 and 9.2 itrid that of type B gelatin is between pH 4.7 and 5.2 [3 11. 1 .l. 10 Methods for measuring bioadhesive stre~~gthof polymers Bioadhesive strength of polymers can be determined using various methods. Most of these methods determine bioadhesion as a factor of separation of the polymer from the mucus srlrfacc. The surface structure of a polymer determines its adhesive properties and not neccssnrily the bulk. Surface analytical studies may include classical preliminary tests such as contact angle measurement or more sophistit \. vcctroscopic techniques, which may be employed to: (a) Study the surface chemistry of the b; adhesive pol) ler. (b) Establish possible interfacial bond:^ ; between the bioatlhesive polymer and mucus. (c) Identify the site of failure of the bit 9 :Iiesive bond [8]. The first method involves the use o ' mioaleter and is based on the Wilhelmy plate niethod which involves surface tension drfsl iiination and consists of a glass plate suspended from a niicrofol-m balance. A glass vial conh:ning the mucus sample is placed on a platform and can be mechanically lowered or elevated. rhis platform is then raised until the plate touches the mucus, the maxin~un~force is recoil :cl in microform and displayed on a recorder when the plate detaches from the mucus gel [19]. Inother technique is the x-ray photoelectron spectroscopy [32]. Application of tensile force as a method of ct.termining bioadhesive strength has been described [8]. This experiment is carried out in a nl ~difiedtensile tester with appropriate cells attached to its vertical bars. The disks or plates are placed between two plates of the cell and a tellsile force applied at constant extension ratio urll the material breaks. The adhesive strength is a function of the maximum elastic modulus, \vhich is measured from the variation of the force with the deformation. Another method involves adsorption or desorption of proteins on or from polymer surfaces. This technique makes use of a thin layer of the test polymer, which is exposed to a solution of a radio-labelled bovine submaxillary mucin [8]. In situ test have become the most desirable method for analysis of the bioadhesive bond strength. Freshly excised mucus membrane is used. An aqueous dispersion prepare with the test polymer is placed on the mucus membrane. A suitable fluid such as distilled water, normal saline, hydrochloric acid; simulated intestinal fluid (SIF) or simulated gastric fluid (SGF) is allowed to flow over the coated beads. In each case, percentage bioadhesion is determined from the amount of adhered material, which is undetached under controlled conditions. The in vivo methods for determination of biondhesion are the most accurate and can either be invasive or non-invasive [8]. It can be said to be the fundamental testing of a bioadhesive formulation since the physiological and especially dietary condition may alter the adhesive properties of polymers. The surface energetics of a polymer may change when the absorption of the drug as well as the polymer surface adsorption of proteins, electrolytes occur through adjacent biological fluid. 1.1.1 1 Applications of bioadhesion The importance of bioadhesive polymers has been recognised for many years, in both medical and pharmaceutical industries. They were first employed in hard tissues and in dentistry where they were used for the adhesion of dentures to gums and in orthopedics as ccments for the adhesion and eventual healing of fractured bones [%I. Bioadhesives have been utilized for artificial replacement of soft tissues. They are routinely used for wound healing instead of sutures in surgery. The ophthalmologists use bioadhesives for the adhesion of the conjunctiva of the eye or upon surgery to put together intraocular lenses with the eye [33]. In recent times, interest have been shown in the use of bioadhesives as controlled release systems for the release of drugs in the buccal or nasal cavity, tlrc intestine or rectum and the urinary bladder [8]. Bioadhesive could be used to target genetically engineered macromo1ecult:s e.g. growth hormone and interferon used in tumors [8]. Bioadhesive polymers are also used as delivery systems for drugs used in the treatment of glaucoma and motion sickness, for contraceptives and narcotic antagonists, for insulin delivery and immunization. Semi-solid mucoadhesive dosage forms have been found suitable for the treatment of mouth ulcers because they can be spread as a thin pellicle over a large portion of the mucosa. 1.2 Dissolu ti011 For a dn~gto be absorbed it must first be dissolved in the fluid at the site of absorption. For example, an orally administered drug in tablet form is not absorbed until drug particles are dissolved or solubilised by the fluids at some point along the gastrointestinal tract, depending on the pH-solubility profile of the drug substance [31]. Dissolution describes the process by which the drug particles dissolve. During dissolution, the drug molecules in the surface layer dissolve to form a saturated solution around tile particles to form the diffusion layer. Dissolved drug molecules then pass tl~roughoutthe dissolving fluid to contact absorbing mucosa and are absorbed. Replenishment of diffusing drug molecules in the diffusing layer is achieved by further drug dissolution and the absorption process continues. If dissolution is fast or the drug is delivered and remains in solution form, the rate of absorption is primarily dependent upon its ability to transverse the absorbing membrane. If, however, drug dissolution is slow due to its physicochemical properties or forn~ulationfactors, the dissolution may be the rate-limiting step in absorption and iiifluence dn~gbioavailability. Any in vitr-o attempt to simulate in vivo conditions cannot be entirely successful because it is not possible to completely reproduce all physiological conditions. 1341. Dissolution rate data when combined with solubility, partition coefficient and pKa results provide an insight to the formulator into the potential in vivo absorption characteristics of a drug. However, in vitro tests only have significance if they can be related to in vivo results. Once such a relationship has been established, in vitro dissolution tests can be used as a quality control test. Nevertheless the importance of dissolution testing has been recognised recently by official compendia with the inclusion in recent editions of dissolution specification using standardized testing procedures for a range of official preparations. 1.2.1 Methods of studying dissolrition 1.2.1.1 Modified USP disintegration apparatus 'This apparatus consists of a basket rack assembly (immersed in an appropriate fluid at 37 " it1 a I-litre beaker) containing six open-ended glass tubes with a 40-mesh stainless steel screen and positioned so that it descends to 1.0 cm from the bottom of the vessel on the downward stroke. Disks are not used in the dissolution procedure [34]. This apparatus is used where the solubility is less than one dosage unit per 100 ml. For granules, the particles either fall through the screen or until a soft mass having no palpably firm core remains on the screen. At appropriate time intervals, samples for analysis are withdrawn from the beaker. 1.2.1.2 The rotating basket assembly This cotlsists of a rotating basket in which the tablet, capsule or granule is placed. The rotating basket is a cylinder 3.6 cm in height and 2.5 cm in diameter. The sides and bottom of the rotating basket are of 40-mesh stainless steel cloth. The rotating basket is attached to a rod, which together are attached to a stirring motor w itls speed-regulating device that may vary the speed from 25 to 200 rpm. In a dissolution test, . ml of dissolution fluid at 37 " is placed in a 1000 mi resin flask fitted with a four-hole cow r. A thermometer is placed in one of the holes of the cover. After a tablet or capsule is place ' in the rotating basket, the stirring rod is passed through the center hole in the flask cover and qtt:lched to the stirring motor. The apparatus is assembled so that the basket is immersed to 1.0 I m from the bottom of the flask. Stirring is then initiated at the specified speed. At apj wpriatc time intervals samples for analysis are withdrawn through the two holes in the covc of the ncl:P. This apparatus is used where the solubility is less than one dosage unit per 100 ' 1.2.1.3 The paddle mixer This consists of a shaft with one or more hor ii ontal arms, which may or may not be pitched. A multiple-arm paddle is known as a gate mixcr If the rpm and the width of the paddle impeller are held constant, the dissolution rate increases as the length of the paddle is increased up to approximately three-fourths the diameter of tJle mixing tank. The usual practice is to have the paddle diameter up to 90 per cent of the diameter of the tank with a ratio of impeller width to impeller diameter of 1 :8 to 1 :12. The dissolution rate is greatest as the paddle impeller approaches the bottom of the mixing tank, and the rate decreases somewhat as the paddle is raised. Proper agitation produces volume cilwlation in all parts of the system. The paddle mixer produces liquid movement tangential to the mixing device and is often accompanied by a swirl with a deep vortex. 1.2.1.4 Rotary impellers Various impellers are available but tli llow developed by them is one of three basic types: axial, radial, or tangential. The marinc type propeller is commonly used for laboratory- scale operations and produces axial flow i.c , fluid is displaced downward along the axis of rotation. The column of liquid below the PI-opellertends to rotate with the propeller without horizontal or vertical flow. The proper u:\e of baffles converts this motion to horizontal and vertical flow. The propeller has a more limited circulating capacity than the turbine or paddle impeller. 'The former uses centripetal force to produce radial flow i.e., fluid is directed outward from the blades toward the impeller tip, with increasing velocity, at right angles to the axis of rotation. For a given set of conditions, the turbine impeller gives a faster dissolution rate than the pacldle or propeller impeller [34]. Most dissolution procedures in pharmacy are accomplished by stirring especially rotational agitation [34]. The intensity of agitation is one of the most important factors in determining the dissolution rate of a solid. I11 general, substances dissolve faster if the system is warmed. If a substance absorbs heat in the dissolution process, its solubility is increased by at1 increase in temperature [34]. The increase in solubility provides an increased concentration gradient. which results in an increased dissolution rate. The increase in temperature increases liiuetic motion and diffusion of the solute tl~roughthe diffusioq layer into the bulk solution, which increases the dissolution rate [34]. 'The solvents for dissolution tests, in order of preference, are: purified water, simulated gastric fluid (SGF) without pepsin, and buffers of pH 4,5, G or 7. A solvent should be selected so that the concentratior drug during the test does not exceed 50 % of saturation in the volunie selected [34]. 1.2.2 Fnctors affecting dissolution ralcs of drug. ;I the gastrointestinal tract 1.2.2.1 Fhysiologic;ll condition 'This considers some factors in the Nr, yes Whitney equation: Eqn. 1 'I'he diffusion coefficient, D, of a drug in [vastrointestinal fluid may be decrcased by the presence of substances, which increasr the 3, 'scosity of the fluids. Hence the presence of food in the GIT may cause a decre,.cse in thc dissolution rate of a drug by virtue of reducing the rate of diffusion of molecules away from the diffusion layer surrounding each undissolved drug part1 :le. 'l'he thickness of fhs diffusion layer, h, will be influenced by the degree of agitation + ;perienced by drug particle in the GIT. I-lence, an increase in gastric andfor intesti~a1 motility may increase the dissolution rate of a sparingly soluble drug by virtue of decreasing the thickness of the diffusion layer around each drug particles. The concentration C, of drug in solution in the bulk of the gastrintestinal fluids will be influenced by such factors as the rate of removal of dissolved drug by absorption through the gastrointestinal/blood barrier and by the volume of fluid available for dissolution. By tl vove equation, a low value of c will favour rapid dissolution of the drug by virtue (icreasing the value of the terms (Cs- C). In the case of drugs whose absorption is dissolution rate limited, the value of C is normally maintained at a very low value by virtue of absorption of the drug. Hence, dissolution occurs under the so-called 'sink' conditions i.e,, under conditions such that the value of (Cs-C) approximates to Cs. Thus for dissolution of a drug in the gastrointestinal fluids under sink conditions, the Noyes-Whitney equation may be expressed as; dm - DACs -- - - ...... Eqn. 2 dt h 1.2.2.2 Physicochemical properties a) Particle size An increase in the total effective surface area of drug in contact with the gastrointestinal fluids will cause an increase in dissolution rate, according to Noyes-Whitney equation [I]. Hence the smaller the particle size, the greater the effective surface area and the higher will be the dissolution rate and bioavailability provided that absorption of the drug is dissolution rate limited [31]. It is possible that particle size reduction may fail to increase the bioavailability exhibited by a dr~~g.This would be the case for a drug whose absorption was not dissolution rate limited. In the case of a poorly soluble, hydrophobic drug, whose absorption is dissolution rate limited, extensive particle size reduction can increase the tendency of the particles to aggregate in the aqueous gastrointestinal fluids with a consequent reduction in effective surface area, dissolution rate and hence bioavailability. b) Solid dispersions The formation of a solid solution of a poorly soluble drug in a water soluble physiologically inert solid should offer a greater improvement in dissolution rate and bioavailability of the poorly soluble drug than would a eutectic mixture (which consists of a microcrystalline dispersion of a poorly soluble e.g. sulphathiazole in a matrix consisting of a physiological inert, readily water-soluble s, cuch as urea). The improved dissolution rate and bioavailability exhibited by drugs pt 1 in this form are contributed by the following factors: i. increased aqueous solubility of the drug due to its extremely small particle size. ii. a possible solubilization effect on the drug by the 'carrier' in the diffusion layer surrounding each dissolving drug particle in the gastrointestinal fluids. iii. a reduction or absence of aggregation and agglomeration of the drug particles exposed to the gastrointestinal fluids. iv. excellent wettability and dispersibility of the exposed drug particles which ensures large effective surface area of the drug in contact with the gastrointestinal fluid. v. possible formation of metastable polymorphic forms (more soluble and rapidly dissolving in gastrointestinal fluids) of the drug during formation of the solid dispersion [31]. c) Crystal form i. Polymorphism: Many drugs can exist in more than one crystalline form, e.g. chloramphenicol palmitate, cortisone acetate, tetracyclines and sulphathiazole. A metastable polymorph usually exhibits a greater aqueous solubility and dissolution rate than the corresponding stable polymorph. Consequently, the metastable polymorphic form of a poorly soluble drug may exhibit an increased bioavailability in comparison to the stable polymorphic foml of that drug. ii. Amorphous solid: Amorphous form of a drug is more soluble and rapidly dissolving than the corresponding c~ystallineform(s), the possibility exists that there will be significant differences in the bioavailabilities exhibited by the amorphous and crystalline fonn(s) of a given poorly soluble drug. c) Complexation The rate and extent of absorption of a drug depends on the effective concentration of that drug. Complexation is one of the principal types of physicochemical interactions, which can influence the effective drug concentration in the gastrointestinal fluids. The other types of interaction are adsorption and micellar solubilization. d) Adsorption The concurrent administration of drugs and medicinal products containing solid adsorbents (e.g. antidiarrhoeal mixtures) may result in the adsorbents interfering with the absorption of such drugs from the gastrointestinal tract. The adsorption of a drug onto solid adsorbents such as kaolin, attapulgite or charcoal may reduce the rate and/or extent of drug absorption from the gastrointestinal tract. e) Chemical stability of drugs in the gastrointestinal fluids Poor bioavailability usually results if a drug undergoes extensive acid or enzyme hydrolysis in the gastrointestinal tract. An alternative method of protecting a susceptible drug from gastric fluid (e.g. Erythromycin) is the pro-drug method of formulation. g) Solubility of the drug in the diffusion layer (salt forms) The dissolution rate of a drug in the gastrointestinal fluids is influenced by the solubility (Cs) that the drug exhibits in the diffusion layer surrounding each dissolving drug particle. In case ofdn~gswhich are weak electrolytes, their overall aqueous solubilities are dependent on pH. The pH in the diffusion layer is not necessarily equal to the pH in the bulk of the gastrointestinal fluids. -c*-, 1.3 Gastroiritestinal motility Many diverse radiological, manometric and radioisotopic tests exist for investigation of gut motility but many are still research tests of limited value in daily clinical practice [35]. Motility of gastrointestinal smooth muscle is affected from the stomach to the colon. Segmental contractions of the smooth muscle mix the intestinal contents. In general, the walls of the viscera are relaxed, and both tone and propulsive movements are diminished. Therefore, gastric emptying time (GET) is prolonged and intestinal transit time is lengthened. Patients with diarrhoea commonly have less spontaneous activity of the sigmoid colon than do people with normal bowel habit and patients with constipation have more. An important factor in diarrhoea may be loss of normal segmenting contractions that delay passage of contents, so that an occasional peristaltic wave may have greater propulsive effect [35]. Diarrhoea due to over dosage with parasympathomimetic agents is readily eliminated, and even that caused by non- autonomic agents can usually be temporarily controlled. However, intestinal "paralysis" induced by antiniuscarinic drugs is temporary; local mechanisms within the enteric nervous system will usually re-establish at least some peristalsis after 1 - 3 days of antimuscarinic drug therapy [36]. Abnormal activity of the gut may cause accelerated, retarded or retrograde (reflux) movement of its contents, with attendant syrnptomatology. Depending on the type of movement, patients may complain of diarrhoea, constipation, colic and a variety of symptoms e.g. nausea, abdominal distension and pain generally classed as non-ulcer dyspepsia [35]. Cliolinergic mechanisms are responsible for modulating motor phenomena in the gut; thus it is not sutprising that cholinomimetic agents such as bethanechol are effective in f. promoting GI motility. Metochlopramide and cisapride arg promoted as more selective motility stimulants (prokinetic agents) as well as erythromycin [36]. Blockade of niuscarinic receptors has dramatic effects on motility and some of the excretory functions of the gut. However, since local hormones and non-cholinergic neurons in the enteric system also niodul~tegastrointestinal function, even complete muscarinic block cannot totally abolish activity in this organ system. As in other tissues, exogenously administered nluscarinic stimulants are more effectively blocked than the effects of parasympathetic (vagal) nerve activily [36]. 1.4 Action or the peripheral nervous system The most obvious effect, which a drug can have on a nerve, is to block the conduction of impulses along it. Usually sensory nerves are blocked more easily than motor nerves, and because drugs block sensation they are called local anesthetics. Any substance, which blocks sensory nerves, however, is likely also to block motor nerves if given in a high enough concentration. 'I'lic effect of drugs can be detected by showing the blockade in conduction electrically, by observing the disappearance of a reflex, or [37] in man, by observing the disappearance of sensation. To study effects in intact preparations the corneal reflex in rabbits and the twitch- response of guinea pig's skin have been widely used. 'These tests can also be performed in man, though the corneal reflex test in man is normally only used qualitatively for demonstrating thc effects of drugs. For the wheal test to be performed in man it is necessary to Cholinergic mechanisms are responsible for modulating motor phenomena in the gut; thus it is riot surprising that cholinomimetic agents such as bethanechol are effective in f. promoting GI motility. Metochlopramide and cisapride promoted as more selective motility stimulants (prokinetic agents) as well as erythromycin [36]. Blockade of muscarinic receptors has dramatic effects on motility and some of the excretory functions of the gut. However, since local hormones and non-cholinergic neurons in the enteric system also modulate gastrointestinal function, even compIete muscarinic block cannot totally abolish activity in this organ system. As in other tissues, exogenously administered muscarinic stimulants are more effectively blocked than the effects of parasympathetic (vagal) nerve activity [36]. 1.4 Action on the peripheral nervous system The n~ostobvious effect, which a drug can have on a nerve, is to block the conduction of impulses along it. Usually sensory nerves are blocked more easily than motor nerves, and because drugs block sensation they are called local anesthetics. Any substance, which blocks sensory nerves, however, is likely also to block motor nerves if given in a high enough concentration. 'I'lic effect of drugs can be detected by showing the blockade in conduction electrically, by observing the disappearance of a reflex, or [37] in man, by observing the disappearance of sensation. 'To study effects in intact preparations the corneal reflex in rabbits and the twitch- response of guinea pig's skin have been widely used. These tests can also be performed in man, though the corneal reflex test in man is normally only used qualitatively for demonstrating thc effects of drugs. For the wheal test to be performed in man it is necessary to use the subject's sensation of pain, instead of the twitch-response of the animal. This method has been used lo obtain accurate quantitative comparisons of local anaesthetics. In the test using the corneal reflex, a solution of the drug is applied directly to the cornea and the lime the corneal reflex disappears is noted. This time is inversely proportional to the rate of onset of anaesthesia. The relative activity of local anesthetics can be estimated by comparing the concentration, which cause the blink reflex to disappear in about the same length of time. The result, however, is only a comparison of the rates of onset of anaesthesia. Like the results obtained with the frog's sciatic plexus these do not indicate differences in the duration or intensity of the effect. With the corneal reflex they are moreover complicated by the failure of certain drugs to cross mucous membranes. With the twitch-response test in a guinea pig's skin, an attempt is made to assess the duration and intensity of the effects, though it ignores differences in the rate of onset and inability to cross mucous surface. 1.5 Medicament From a therapeutic point of view, drugs to be formulated into a sustained release product should possess one or more of the following properties [38]: - should be used for chronic disease states. - have regular absorption - have short plasma half life. - have no serious side effects when produced in the sustained release form. Sustained release dosage forms offer the advantage of convenience, maintenance of more uniform concentration in blood and other body fluids and elimination of side effects that may result from too rapid release of the drugs [38]. Because they are usually taken over a twelve-hour period, they also reduce the likelihood of missed doses and increase the patient's compliance to the drug regimen. 1 S.1 Chlorpropamide Chlorpropamide is a white, crystalline powder, practically insoluble in water, freely soluble in acetone and in methylene chloride, soluble in alcohol. It dissolves in dilute solutions of alkali hydroxides. It shows polymorphism [39]. Chlorpropamide is a sulphonyl urea and is an orally active hypoglycaemic agent, which reduces the blood sugar concentration. It probably acts by stimulating insulin secretion, as it has no action on muscle-glucose metabolism when given alone. It is effective only in the presence of functioning islet tissue. It is readily absorbed from the gastrointestinal tract and is extensively bound to plasma proteins. The half-life in plasma is about 35 hours. About 80 to 90 % of a dose is excreted, partly unchanged, in the urine within 4 days [36]. Chlorpropamide is indicated for the treatment of mild to moderately severe uncomplicated maturity onset diabetes mellitus unresponsive to diet alone but it is unsuitable for diabetic patients with ketonuria or diabetic ketosis. Chlorpropamide is also used in patients to treat partial central diabetic insipidus. The side effects include skin sensitization, gastrointestinal disturbances, leucopenia, intolerance to alcohol and jaundice. In the event of sore throat or fever, repeated white cell counts should be carried out because abnormalities in the circulating blood may not appear for several days. It is contra-indicated in diabetes mellitus complicated by fever, trauma or gangrene and in patients with impaired renal or hepatic function or serious impairment of thyroid or adrenal functions. It is teratogenic and feticidal. It is excreted in breast milk, so use by nursing mothers may cause hypoglycaemia in the infant WI. A disulfiram-like reaction may occur in patients taking alcohol, during treatment with chlorpropamide. Hypoglycaemic effects of chlorpropamide may be enhanced by chloramphenicol, clofibrate or halofenate, cyclophosphamide, dicoumarol, MA0 inhibitors, phenylbutazone, propranolol, and other beta-adrenergic blocking agents and some sulphonamides. Adrenaline, corticosteroids and diuretics may diminish the hypoglycaemic effects. Propranolol may mask symptoms of hypoglycaemia. An overdose of chlorpropamide can produce hypoglycaemia. Mild hypoglycaemic symptoms without loss of consciousness or neurologic findings should be treated aggressively with oral glucose and adjustments in drug dosage andlor meal patterns. Severe hypoglycaemic reactions with coma, seizure or other neurological impairment occur infrequently, but constitute medical emergencies requiring immediate hospitalization. If hypoglycaemic coma is diagnosed or suspected, the patient should be given rapid intravenous injection of concentrated (50 %) glucose solution. This should be followed by a continuous infusion of a more dilute (10 %) glucose solution at a rate that will maintain the blood glucose at a level above 100 mgldl. Patients should be closely monitored for a minimum of 24 to 48 h since hypoglycaemia may recur after apparent clinical recovery [36). 1.6 Viscosity, rheology and the flow of fluids The viscosity of a fluid may be described simply as its resistance to flow or movement. Thus, water which is easier to stir than syrup is said to have the lower viscosity [3 11. Rheology may be defined as the study of the flow properties of materials. The importance is to merely characterize and classify fluids and semi-solids. For instance, in the British Pharmacopoeia [40], substances such as liquid paraffin have been controlled by a viscosity standard for many years, whereas a yield value test for Carbomer gels has only been introduced more recently. However, the development and adoption of dissolution testing has given added importance to knowledge of solution viscosity since it may enable mechanisms of dissdution and absorption to be determined. Furthermore, advances in the methods of evaluation of the viscoelastic properties of semi-solids and biological materials have produced useful correlations with bioavailability and function [3 11. The need for a proper understanding of the rheological properties of pharmaceutical material is an essential fundamental to the preparation, development and evaluation of pharmaceutical dosage forms. 1.6.1 Newtonian fluids - viscosity values for Newtonian fluids 1.6.1.1 Dynamic viscosity Newton put up a quantitative basis that the rate of flow (D) was directly related'to the applied stress (T): the constant of proportionality is the coefficient of dynamic viscosity (q) more usually referred to simply as the viscosity. Simple fluids which obey the relationship are referred to as Newtonian fluids, and fluids, which deviate are known as non-Newtonian fluids. T=~D ...... Eqn.3a ...... Eqn. 3b The kinematic viscosity (V) also characterizes a fluid and is defined as the dynamic viscosity ,I (q) divided by the density of the fluid (p): '1 v=- ... , ...... Eqn. 4 P Relative and Specific viscosities The relative viscosity or viscosity ratio (qr) as given by Eqn. 5. q r 2- ...... Eqn. 5 rl, and the specific viscosity is given by Eqn. 6. qsp=qr-1 ...... Eqn. G The solvent in pharmaceutical products is normally water. For a colloidal dispersion, the equation derived by Einstein may be used to study the viscosity. q = qo(1+2.5$) .a...... Eqn. 7a where $ is the volume fraction of the colloidal phase (the volume of the dispersed phase divided by the total volume of the dispersion). The Einstein equation may be rewritten as: '1 -- =1+2.5$ ...... Eqn. 7b 'lo 4' Since - is equal to the relative viscosity, then it can also be rewritten as 'lo 11 '7-'lo - -- - 1 = -- -2.5 $ ...... Eqn. 8 'lo 'lo so that the left hand side equals the specific viscosity. Rearranging it gives: and since the volume fraction will be directly related to concentration, then it can be rewritten as: Eqn. 10 when the dispersed phase is a high molecular mass polymer then a colloidal solution will result and provided moderate concentrations are used then it can be expressed as a power series. . . ,.. ... Eqn. 11 Intrinsic viscosity 1f5,the viscosity number or reduced viscosity is determined at a range of polymer C concentrations and plotted as a function of concentration, then a linear relationship should be obtained and the intercepts produced on extrapolation of the line to the ordinate will yield the constant KI , which is referred to as the limiting viscosity number or the intrinsic viscosity, [q], when the units of concentration is in g dl-'. The limiting viscosity number may be used to determine the approximate molecular mass (M) of polymers using Mark-Houwink equation: hl = KMa ...... Eqn. 12 where K and a are constants that must be obtained at a given temperature for the specific polymer-solvent system. Also, the value of the two constants provide an indication of the shape of the molecule in solution; spherical molecules yield values of a = 0 whilst extended rods have values of greater than 1 .O. A randomly coiled molecule will yield an intermediate value (z0.5). 1.6.2 Non-Newtonian fluids Most pharmaceutical fluids do not follow Newtonian law because the viscosity of the fluid varies with the rate of shear [3l]. The reason for these deviations is that the fluids concerned are not simple fluids like water and syrup but are disperse or colloidal systems including suspensions, emulsions and gels. These materials are known as non-Newtonian and with the increasing use of sophisticated polymer-based delivery systems more examples of such behaviour are found in pharmacy. 1.6.2.1 Types of non-Newtonian behaviour More than one type of deviation from Newton's law can be recognized and it is the type of deviation, which occurs that can be used to classify the particular material. 1. Plastic (or Bingham) flow A plastic material does not flow until a yield value of shear stress has been exceeded and at lower stresses the substance behaves as a solid (elastic) material. This type of flow is exhibited by concentrated suspensions particularly if the continuous phase is of high viscosity or if the particles are flocculated. 2. Pseudoplastic flow A pseudoplastic material flows as soon as a shear stress is applied. Materials exhibiting this behaviour are said to be pseudoplastic and no single value of viscosity can be considered as characteristic. The viscosity can only be calculated fiom the reciprocal of the slope of a tangent drawn to the curve at a specific point. Such viscosities are known as apparent viscosities (qa,,) and are only of any use if quoted in conjunction with the shear rate at which the determination was made. Since it would need several apparent viscosities to characterize a pseudoplastic matcrial then the most satisfactory representation of the material is by means of the entire flow curve. However, it is frequently noted that at higher shear stresses the flow curve tends towards linearity indicating that a minimum viscosity has been attained. When this is the case then such a viscosity can be a useful means of classification. Such materials are most usually typified by aqueous dispersions of hydrocolloids such as tragacanth, acacia, alginates, methylcellulose, gelatin, SCMC and synthetic materials such as polyvinylpyrrolidone (PVI'). The presence of long high molecular weight molecules in solution results in entanglement together with association of immobilized solvent. Under the influence of shear the molecules tend to become disentangled and align themselves in the direction of flow and this together with the release of some of the entrapped water accounts for the lower viscosity. At any particular shear rate equilibrium will be established between the shearing force and the re-entanglement brought about by Brownian motion. 3. Dilatant flow Dilatant materials show viscosity increase with increase in shear rate. Since such materials increase in volume during shearing then they are referred to as dilatant and exhibit shear thickening. It is less common than plastic or pseudoplastic but may be exhibited by dispersions containing a high concentration (x 50 %) of small deflocculated particles. Dilatancy can be a problem during the processing of dispersions and granulation of tablet masses when high speed blenders and mills are employed. 1.6.3 Methods used in determining the fF yoperties of simple fluids A wide range of instrument exists. which may be used to determine the flow properties of Newtonian fluids. It would be impossible to review all the instruments, which could be used to measwe viscosity and consequently, this is limited to those instrument, which are mentioned in the British Pharmacopoeia (BP) 1980 [40] together with others, which are commonly used for simple fluids and dilute colloidal solutions. Capillary viscometers examples Ostwald U-tube viscometer and suspended level viscometer; Falling sphere viscometer. Determination of the flow properties of Non-Newtonian fluids With such a wide range of rheological behaviour it is extremely important to carry out measurements, which will produce meaningful results. It is crucial therefore not to use a determination of viscosity at one shear rate (such as would be for a Newtonian fluid) since it could lead to completely erroneous comparative results. Simple point determinations are probably an extreme example but are used to emphasize the importance of properly designed experiments. A number of methods are used to determine the flow properties of non-Newtonian fluids among which are the use of rotational viscometers such as concentric cylinder and cone plate viscometers. 1.7 Gel permeation with cross-linked dextraus (SephadexTM) Gel filtration first became an established laboratory technique with the introduction of cross-linked dextrans in 1959. Since then, gel filtration has been increasingly used both in analytical and in preparative work, as well as on a production scale in the chemical industry. Before cross-linked dextrans were introduced, fractionation and separation of molecules according to size could only be carried out by very time-consuming or expensive methods. Using the appropriate type and grade of cross-linked dextrans, such separations can now be performed rapidly and simply. They are particularly useful for chromatography of substances of biological origin, which are often very labile. A gel filtration experiment can be schematically described in the following way. Molecules larger than the largest pores of the swollen cross-linked dexlrans i.e. ahove the exclusion limit cannot penetrate the gel particles and therefore, they pass through the bed in the liquid phase outside the particles. They are thus eluted first. Smaller molecules, however, penetrate the gel particles to a varying extent depending on their size and shape. Molecules are therefore eluted from a cross-linked dextran bed in the order of decreasing molecular size. The dextran n~acromoleculesare cross-linked to give a three-dimensional network of polysaccharide chains. Because of its high content of hydroxyl groups, it is strongly hydrophilic and the cross-linked dextran beads swell considerably in water and electrolyte solutions. Various cross-linked dextran types are available, differing in their swelling properties. The degree of swelling is an important characteristic of the gel. Gels in which the matrix is a minor component are used for fractionation of high molecular weight substances, whereas the compact gels are used for work with low molecular weight compounds. 1.7.1 Cross-linked dextran properties 1.7.1.1 Physical properties The cross-linked dextrans of the G-series differ in degree of cross-linkage and thus in swelling properties. The water regain value is used to characterize the gels with respect to their swelling ability. It represents the amount of water imbibed by the gel grains on swelling and does not include the water between the grains. The type numbers refer to the water regain value of the gels. Cross-linked dextran G-10 thus has a water regain value of 1 and that of G- 200 has a water regain of 20. Cross-linked dextrans of the G-series also swell in dimethylsulphoxide, forrnamide and glycol. Mixtures of water and the lower alcohols may also be used. In addition to these solvents, the G-10 and G-15 swell in dimethylformamide. It should however be noted that the solvent regain in solvents other than water is not equivalent to the water regain. Cross-linked dextrans of the LH-series are an alkylated product with lipophilic as well as hydrophilic properties. Therefore swells in many organic solvents as well as in water. Cross-linked dextrans are available in different particle size grades. The superfine grade is intended for column chromatography requiring very high resolution and for thin layer chromatography. The fine grade is recommended for preparative purposes, where the extremely good resolution that can be achieved with the superfine grade is not required, but where the flow rate is of greater importance. The coarse and medium grades are intended for preparative chromatographic processes where a l~ighflow rate at a low operating pressure is essential. In addition the coarse grade is suitable for batch procedures. 1.7.1.2 C31emicalstability of cross-linked dertrans The cross-linked dextral) gels are insoluble in all solvents (unless they are chemically degraded). They are stable in water, salt solutions and organic solvents, alkaline and weakly acidic solutions. In strong acids the glycosidic linkages in the gel matrix are hydrolysed. However, cross-linked dextrans can be exposed to 0.1 M HCI for 1-2 hours without noticeable effects, and ill 0.02 M HCI they are stiIl unaffected after G months. Prolonged exposure to oxidizing agents will affect the gel and should be avoided. They can be sterilized in the wet state by autoclaving for 40 tnins at 110 'C without any changes in the properties of the gel. If dry they are heated to more than 120 "C, it will start to caramelize. 1.7.2 Applications of cross-linked dextrans 1.7.2.1 Analysis of multi-component samples In gel filtration, quantitative yields are obtained, as there is no irreversible retention of small amounts of substances on the columns. This makes cross-linked dextrans very usefi.11 for analytical purposes. The chromatographic experiments can be made quantitative by recording continuously some physical variable in the effluent (e.g. light absorption, optical activity, refractive index). The effluent can also be collected in fractions and the amounts of substance in them evaluated by physical, chemical or biological tests. Gel permeation has often been used for analysis where few or no other methods are available. It is thus used for analysis of protein mixtures and fluids of biological origin. In clinical laboratories, gel filtration is now a standard method for diagnosis of proteinaemias such as Waldenstr6m's disease [4 1-44]. 1.7.2.2 Determination of molecular weights One of the most striking properties of cross-linked dextran gels as chromatographic materials is their capacity for separating substances according to molecular size. For proteins, extensive investigations have shown that the elution volumes of globular proteins are largely determined by their molecular weights 145-501. Over a considerable range, the elution volume is approximately a linear function of the logarithm of the molecular weight [48, 501. This method can also be used for determination of molecular weights of peptides and of other n~acromoleculesthan proteins [51, 521. As the relationship between molecular weight and elution volume is different for different types of molecules, a separate calibration curve should be determined for each type. With this method the molecular weight of enzymes can be determined even in crude preparations [53-551. The enzyme activity in the colunin effluent is measured and thus the elution volume can be determined. From the calibration curve, produced by chromatography of proteins of known molecular weight, the molecular weight of the enzyme can then be estimated. This is the only method of estimating molecular weight of proteins, which does not require very extensive purification. 1.7.2.3 Zone electrophoresis Cross-linked dextrans can be used in zone electrophoresis as a supporting material in vertical columns as well as in horizontal troughs and on thin-layer plates. In electrophoresis, they have the advantage of giving sharp and well-defined zones. The material exhibits low electroosinotic flow and negligible protein adsorption and is also very easy to handle [56 - 581. 1.8 Muciris Over the past 5 - 10 years several studies carried out on mucus glycoproteins from many organs havc suggested that these macromolecules consist of sub-units held together by interchain disulphide bonds and further stabilized by non-covalent interactions 1591. The end result of nlilltiple interconnections is an extended and random gel network, which imparts to mucus secretions their characteristic property of visco-elasticity. Evidence that disulphide bonds (S-S) play an important stl-uctural role has been provided by demonstrations that thiol- group (-SH) reagents decrease the viscosity and increase the solubility of native niucils secretions [59], and in some cases decreases the molecular weight of purified mucins. For example, the sedimentation coefficient of a large porcine gastric mucin decreased from 33 s (mol. wt. 2.3 x 10" to 14 s (mol. wt. 2.5 x 10.5) after treatment with 0.2 M of 2-mercaptoethanol [59]. Somewhat similar but less dramatic effects have been observed after reduction of bronchial mucus glycoproteins 1591, cervical mucins [59, 601 and egg white R-ovornucin by the same reagent. Despite these observations, there remain some difficulties in accepting a central role for S-S bonds in the molecular architecture of all mucin molecules. Firstly, highly purified mucins characteristically have a very low content of cysteine. In purified bovine submaxillary mucin, here are no cysteine residues at all 1591, and no evidence of dissociation by-SH agents [59). I11 bronchial mucins, the disulphide bridges cleaved by reducing agents belong mainly to the protein fraction of the mucus rather than the gI; copvo~u~nfraction. One explanation of these anomalies is that mucins may normally be polymer^ :ed through specific cross-linking peptidcs, which are enriched in cysteine and form covalent disulphide bonds with much cysteine residues. 'l'he linking peptides are tho~ghtto exist eithc as specialized non-glycosylated areas of the peptide core of lnucin glycoproteins [59], or as disc! te peptides, which intertal~glewith glycosylated inucin fibres and form S-S bonds with them [59] Another possibility that should be considered is that some nlucin or glycopeptide molecules, be( wetheir three dimensional structure is stabilized by intramolecular S-S bonds, are able to in! lrpenetrate each others' molecular domains, producing secondary strong non-covalent cross-linkinp or aggregation into mrtciti polymers. 1.8.1 The biological mrrcus membrane Mucus is a translucent and vicid secretion, which forms a thin, continuous gel blanket adherent to the mucosal epithelial surface. It protects the lining of the hollow organs in contact with external media. It is secreted by the goblet cells lining the epithelia or by special exocrine glands. The composition varies with species, anatomical location and the pathological state but its general composition inclucles [6 I]: Water ...... 95 % Free proteins ... . . , ...... 0.5 - 1 % Minerals ...... 1 % Glycoproteins and Lipids ...... 0.5 -5 % A glycoprotein comprises of a protein core with a carbohydrate side chain covalently attached to it. The side chains contain sialic acid, sulphuric acid residues or L-fucose group at the end of the chains. The presence of sialic acid with pKa 2.6 gives the mucus its dense negative charge, which at physiologic pH contributes significantly to bioadhesion [62]. The basic a~ninoacids of the protein component are serine and threonine. The glycoproteins are responsible for gel-like characteristics believed to be the major structure-fonning component of mucus giving rise to cohesive and viscoelastic nature of the mucus gel or layers held together by disulfide bonds and electrostatic and hydrophilic interactions making gels appear as highly entangled ~nacro~nolecularchains. The main functions of the mucus layer are protection, barrier, adhesion and lubrication [63], and in the case of snails, directional orientation. Altl~oughsnails can move over amazingly great distances, snails often have their preferred habitat range to some extent, they might even be able to orientate themselves within this habitat, knowing where to find food and where to find shelter. Garden snails, for example, use to rest in sheltered places during hot summer days, and at night, they move towards cultivated plants to enjoy their meal. Some snails indeed use to go back to the same places where they came from and probably they use their own mucus trails as an aid in orientation C641. Homing is even more crucial for survival in limpet snails (Patellidae or Acmaeidae) living on rocky marine shores. Every limpet occupies his very own place on the rock, and while growing up; it adheres so firmly to the ground that its shell adapts to all irregularities in the rock structure. That means, the shell grows so that it completely covers the animal on an uneven piece of rod, and isolates it hermetically from its environment. No single water could enter the space between the shell and the rock, and in times of strong wave action or other threats, the animal can keep itself tight on the ground with a sucking force of about 15 kg [64]. 'This hermetic slieltesing is particularly important in times of low tide, when the marine shore snails are exposed to intense sun. But still, limpet snails have to move around from time to time in order to find suitable place to grazc for food. They do this at times of high tide when they are submerged, given that the waves are calm enough. Under such calm conditions, each limpet starts to wander around, moving in a small circle up to a distance of about 1 m. It seems that the snail miss their home place only extremely rarely, although suitable rocks are mostly inhabited by a considerable density of limpet individuals. This is because limpets mark their personal place, as weli as their mucus trail around it, with certain chemical substances. These cheniical markers are very stable and won't he washed away easily by the sea. Each individual follows only its own trace and tends to avoid the traces of other individuals. Mucus trail following seems to occur amazingly frequently in snails. However, it does not only seem to be of importance in homing and orientation. By following mucus trails of conspecifics, snails do also find mating partners or, in snail species which tend to live in social aggregations, a group of conspecifics. And, less nicely, some predatory snails also follow the mucus trails of their prey species. 1.8.2 Characterization of mucin Because of the heterogeneous nature of the natural mucus secretions, many mode1 systems have bee11 suggested for use. Knowledge of the chemistry of membrane glycoproteins is limited, largely because of difficulties with solubilization and isolation. The secretions of the digestive tract contain a great number of glycoproteins [65]. N- acetylgalactosainine, N-acetylglucosamine, galactose, glucose, mannose, fucose and sialic acid were identified as carbohydrate components of human gastric juice [59, 661. Several glycoproteins isolated from digestive-tract secretions were high molecular weight substances containing four major carbohydrate residues: N-acetylgalactosamine, N-acetylglucosamine, galactose and fucose [65, 673. The glycoprotein from pig gastric mucus, of molecular weight 2 x lo6 Daltons, consists in average of four glycoprotein subunits of molecular weight 5 x 10' Daltons and is dissociated on digestion with proteolytic enzymes [67] or by reduction of disulphide bridges [68, 691. A carbohydrate-free protein of molecular weight 70,000 Daltons joined to the glycoprotein by disulphide bridges, and probably having a central structural role, is also released or digested on reduction or proteolysis respectively of the pure gastric mucus glycoprotein [69]. Human gastric mucus glycoprotein is the same size and has the same type of polymeric structure as the glycoprotein from the pig gastric mucus 1701. There is evidence for the importance of disulphide bridges in the structure of glycoproteins in other mucus secretions of the gastrointestinal tract, respiratory tract [69], the cervix [71] and the ovarian cyst [72]. However, the polymeric structure and location of the disulphide bridges in these glycoproteins remains unclear, principally because of the difficulties in isolating the undegraded glycoprotein in a soluble form and free of non-covalently bound protein or non-covalent intermolecular polymerizations. There are reports, too, of mucus glycoproteins that are not dissociated by reduction of disulphide bridges, e.g. sheep submaxillary-gland mucus, which is a polymer formed by strong non-covalent interactions between subunits of molecular weight 1.54 x 10" Daltons that are dissociated only in high salt concentrations [59]. Also, rat small intestinal 43 mucus [73J and hr~mansmall-intestinal mucus [73], are not dissociated by reduction with 10 niM -- dithiothreitol in 6 M guanidinium chloride, 14owevcr, it is clearly important to obtain a better appreciation of the extent and nature of the macromolecular structures of these mucus glycoproteins, particularly since the integrity of their polymeric fotm is essential for the gel--forming properties of mucus [G9, 731. Numerous analytical neth hods are employed in characterization of mucins e.g. Equilibrium density-gradient centrifugation in caesium chloride, gel filtration chromatography, other cllromatograpliic mcthods [73] and physical measurements [74]. 1.8.3 Intel-action of much with biological compounds Gastrointestinal mucus of inammals and other vertebrates is a viscous secretion lining the intestinal tract, and composed chiefly of high molecular-weight mucin glycoproteins [59, 69, 721. Until recently, it has been assumed that mucus is an inert blanket, serving as a mechanical barrier against potentially injurious chemicals, bacteria and enzymes [73]. In recent years, however, it has become apparent that much glycoproteins are capable of interacting in various ways with many biologically important compounds such as enzymes, cations, drugs, viruses, cell surfaces and bacteria [73]. Some of these interactions alter mucus structure, solubility, permeability and influence the protective functions of mucus [73]. Interactions of mucin with other proteins have not been investigated systematically 1711. Such interactions, if they exist, could affect the properties of mucus in both physiological and pathological states since it is known that the quantity of non-mucin proteins within mucus is subject to considerable variation. For example, the non-much protein content of cervical mucus varies during the oestrous cycle, and it has been suggested that the hormone-associated changes in the consistency of cervical mucus may rely on local secretion of proteins [73]. Mucus obtained from the respiratory, reproductive and intestinal tracts of patients with cystic fibrosis is characterized by its thick, viscous and sticky quality. The mucus is also highly enriched in protein, particularly albumin [73, 741. Bronchial glycoproteins are intimately associated with non-covalently bound proteins, suggesting that some positive interaction between these molecules may be taking place [59]. 1.8.4 A new glycosaminoglycan from the giant African snail Acltafinafulica A new glycosaminoglycan has been isolated from the giant African snail Achatina jidica. (Fig. 1) This polysaccharide has a molecular weight of 29,000 Daltons calculated based 011 the viscometry, and a uniform repeating disaccharide structure of (1 -+ 4)-2-acetyl,2-deoxy- a-D-glucopyranose (1-+4)-2-sulfo-a-L-idopyrailosyluronicacid (1-+4). This polysaccharide represents a new, previously undescribed glycosaminoglycan. Glycosamino-glycans (GAGs) are a family of linear anionic polysaccharides that are typically isolated as proteoglycans linked to a protein corc. 'The biological functions of proteoglycans, including the regulation of cell growth, result, in large part, through the interaction of the GAG chains in proteoglycans with proteins, such as growth factors and their receptors [75]. There are two major classes of GAGs: 1. Glycosaminoglycans, including heparin, heparan sulphate, hyaluronic acid, and keratan sulfate; and 2. Galactosaminoglycans, including chondriotin and dermatan sulphates [75]. It is related to the heparin and heparan sulphate families of glycosaminoglycans but is distinctly different from all known members of these classes of glycosan~inoglycans. Heparin and heparan sulphate have been the subject of intensive study because of their well recognized ability to bind many different proteins that regulate a variety of important biological processes 1761. Heparin and heparan sulphate GAGS are comprised of alternating I -+ 4 [inked glycosamine and uronic acid residues. Meparan sulphate is con~posed primarily of monosulphated disaccharides of N-acetyl-D-glycosamine and D-glucuronic acid, while heparin is composed mainly of trisulphated disaccharides of N-sulfoyl-D-glucosamine and L-iduronic acid [76]. GAGS have been isolated fiom various tissues obtained from a large number of animal species including both vertebrates and invertebrates [75, 771. An exhaustive assessnlent showed that while a large number of invertebrate species contain GAGS,molluscs are a particularly rich source of these sulfated polysaccharides [77]. Invertebrates were first shown by Burson et al 175 J to contain a heparin or heparan sulphate type GAG. Heparin has only been found in one invertebrate phylum, the mollusc, and it often corresponds to up to 90 % of the total GAG content of these organisms. While the heparins isolated from various molluscs are structurally different from human heparin and pharmaceutical heparins 1751, mollusc heparins contain antithrombin-dependent anticoagulant activity associated with the presence of the unique 3-0-sulphated glucosamine residue found in the antithrombin pentasaccharide binding site commou to all anticoagulant heparins [75]. The structure of this polysaccharide, with adjacent N-acetylglucosamine and 2-sulpho- iduronic acid residues, also poses interesting questions about how it is made in light of our current understanding of the biosynthesis of heparin and heparan sulphate. Snails' glycosaminoglycan represents 3 - 5 ?h of the dry weight of snail's soft body tissues, suggesting important biological roles for the survival of this organism, and may offer new means of control of this pest. Snail glycosaminoglycan tightly binds divalent cations, such as copper (11), suggesting a primary role in metal uptake in the snail. Finally, this new polysaccharide from snails might be applied, like the Escherichia coli K5 capsular polysaccharide, to the study of 'OSO, - 1 n=54 Scheme 1. Structure of snail GAG glycosaminoglycan biosynthesis and to the semisynthesis of new glycosaminoglycan analogs having important biological activities. The large amount of this GAG found in snail also raises some interesting questions about its biological function(s). Many roles can be proposed for this GAG including: 1) binding, uptake, and transport of divalent cation; 2) an anti-desiccant; 3) a molecule linked to molecde 1751. The most likely role of snail GAG is its involvement in cation binding. A. fulicci is a very large gastropod that requires substantial quantities of calcium for its shell 1751. In addition, other divalent ions are critical components of their diets. The blood of snails is blue, as hemocyanin is the copper-based carrier of oxygen in these animals 1751. This shows that snail GAG binds copper (11) much more tightly than heparan sulphates and with about the same avidity of heparin, which has a 3-fold higher level of sulphation. GAGS are known to organize and hold water [75]. Since snails are particularly prone to dehydration, this suggests a \+. second role for this polysaccharide. Snails move on mucus slime through wave-like undulations of their foot muscle [75]. This high molecular polysaccharide is extremely viscous and may represent a component of this slime. Antibiotic properties have been reported for heparin [75], and A. fulicn is known to make a bactericidal glycoprotein that is found in its mucus [75], suggesting that snail GAG may have a protective role. Other interesting questions about the snail GAG is that chemical modification of snail polysaccharide using relatively simple methods, i.e. de-N-acetylation and re-N-sulphation, should lead to a structurally homogenous polysaccharide with the minimum structural features for binding fibroblast growth factors [75]. Finally, the giant African snail is considered a major pest in many parts of the world [75]. 'I'he use of heparin lyase 11 or I;. heparinum (a soil isolate) capable of degrading its major polysaccharide might provide a biological means for controlling A.fulica [75]. 1.9 Snails Snails have been eaten since prehistoric times. Roasted snail shells have been found in archaeological excavation [64]. In ancient Rome, there were "cochlearia" gardens where snails were fattened up before being eaten. The Romans selected the best snails for breeding. Snails were eaten during Lent (Christians' fasting period) on the continent. In a few places, large quantities of snails were consumed at Mardicras or carnivals, as a foretaste of Lent (Christians' fasting period). Edible snails also played a role in folk medicine, and recent study has shown that glandular substances from the edible snails cause agglutination of certain bacteria, and therefore could be of value against whooping cough and some other diseases [64]. Snails also host parasites and disease organisms. However, there are thousands of varieties of land snails, ranging in size from very tiny ones about one millimeter long to the giant African snail, which occasionally grows up to a foot long. "Escargot" most commonly refers to either Iielix aspersa or to Helix pomafia, although some other varieties of snails are eaten, and the giant African snail, Acltntinafulica, is sliced and canned and sold to some consumers as escargot [64]. Helix aspersu, the French "petit gris", or "small grey snail", the "escargot chagrine", "la zigrinata", measures 30 to 35 mm across the shell when mature. The shell of a mature adult has 4 or 5 whorls and is a native of the shores of the Mediterranean and up the coast of Spain and France. It is found on most of the British Isles, where the Romans introduced it in the first century AD. This variety was introduced into California by French immigrants and has become a serious pest. It has also been introduced into a number of Eastern and Gulf states even before 1850 as well as in South Africa, Mexico, Argentina and New Zealand. It has a life span of 2 -- 5 years and is more adaptable to different climates and conditions than many snails, and is found in woods, fields, sand dunes, and gardens. Their adaptability not only increases aspersa's range, it makes farming aspersa easier and less risky [64]. Helix pomntia, the "Roman snail", "apple snail", "luna", "La vignaiola", the German "Weinbergschnecke", the French "escargot de Bourgogne" or Burgundy snail or "gros blanc", measures about 45 inm across the shell. It is native over a large part of Europe and lives in wooded mountains and valleys up to about 6,000 feet (2,000 m) altitude as well as in vineyards and gardens. It was introduced into Britain by Romans and into U.S. in Michigan and Wisconsin by imnligrants. Helix promatia is preferred by many over Helix aspersa for its flavour and its larger size, as the "escargot par excellence". Otda lacteal or Helix lacteal is popular with Italians, and is sometimes called the . "vineyard snail", "milk snail", and "Spanish snail". It has a white shell with reddish brown spiral bands, which is about an inch to 35 mm in diameter. Considered better tasting than aspersa by many. This snail has been established in California, Georgia and a few other states. Cepaea nentoralis, or Helix nemoralis, the "wood snail", the Spanish "vagueta" measures about 25 mm across the shell and inhabits central Europe and has been introduced and established in a number of places. Achntina fulica, the giant African snail, grows up to 1 foot long, overall body length. This snail was purposely introduced into India in 1847. An unsuccessful attempt was made to establish it in Japan in 1925. It has been transported to other Pacific locations both on purpose and accidentally and is now a serious pest in many places. It has been introduced into the U.S. around Miami and Hollywood and in Hawaii, where it not only causes crop damage, but, due to their large size, it is nuisance due to the slime and fecal material and the smell when something like bait causes large numbers to die. Considerable effort has been made to eradicate Achatina [64]. Others see the same snails that some persons raise or gather as food as a major pest. Introduced slug and snail varieties tend to be worse pests than native species, probably due in part to the lack of natural controls [64]. California alone is reported to spend $37 million fighting snail pest that attack crops. Crops affected range from leafy vegetables to fruits, which grow near the ground, such as strawberries and tomatoes, to citrus fruits high up on trees. Since large infestations of snails can do devastating damage, many states have quarantines against nursery products and some other products from the farm. Snail as a gastropod moves on a flat sole, which in many species has a large pedal gland. This gland secretes mucus onto the surface over which the sole moves. Locomotion is achieved by waves of muscular contractions that pass down the foot. 1.9.1 The giant land snails of West Africa There is no such thing as the giant African or West African snail since there are many genera containing numerous species and the term can be interpreted in various ways by people from different countries or regions [64]. Such different interpretation may be due in part to the preponderance of a particular species in an area and the preference for it over others as food. For instance, the giant snail in Ghana is taken to mean Achatina achatina (Linnk), but in Nigeria this might refer to Archachatina nzarginata (Swainson) and in East Africa to Achatina firlica Bowdich [78]. There are, therefore, several giant land snails in Africa, and not just one species. In West Africa, the edible giant land snails belong to two genera, namely: Achatina lamarck and Archachntina alhers. The two most important ckissical monographs on the giant land snails of Africa are concerned with comprehensive revision of the classification of the group, and on the anatomy [79, 801 respectively. This comprehensive classification suffers from the fact that it depended solely upon shell characters. On the genus Achatina, some workers have made important discoveries [8 1- 841, while notable work on the genus Archachatina has been documented [85 - 891. The two genera are not considered pests in West Africa because of the high indiscriminate human predation rate, especially during the main rainy season of the year. The iniportance of snails in the diet of the forest-dwelling people of West Africa is well known. Thus a survey shows that snail meat ranks high among the meat preferences of the Ashanti in Ghana 1901. However, the seasonality of the supply of snail limits their use for meat on a continuing basis. Since there are no organized snail farms in West Africa, there is therefore a need for large-scale farming of snails Tor use as food. The genus Achatina has shell broadly ovate and subglobular with regular conical spine and narrow apex. The egg production is numerous and usually of small sized. In the female reproductive tract, the vagina is tubular and long and the spermathecal duct is extremely short. The major difference in Archachatina is that the shell is broadly ovate and subglobular with wide, bulbous or dome-shaped apex; production of few and relatively large eggs and in the female reproductive tract, the vagina is tubular and short and the spermathecal duct is very long and slender. 1.9.2 The genus Aclratiiia The genus Achatina is found in continental Africa South of the Sahara. Its northern limits are the Gambia (14 ON) in the West and the region '0 Lake Chad in the East, while the southern limit extends to the Orangc River in South Africa. The genus occurs in East Africa and has been introduced to various countries of the Pacific Ocean. The estimated number of valid species of Achatina is at between 65 and 80 and eight subgenera of all the species occurring in Africa were created [79]. The subgenus Achatina is the major one found in West Africa or the Uppcr Guinea zone, stretching from Senegal to Mount Cameroon. 'The furthest it cxtends inland is in Liberia (some 290 km from the coast) and some 160 km from the coast in Ghana [82]. In the wild state, it shows a predilection for decaying vegetable matter and it is often coprophagus. When farm-reared, the snail thrives on specially prepared leaves of the wild lettuce (Lettuce taraxacifolia), various other leaves and on ripe fruits [84, 911. The West African species of Achatina are true forest species. They prefer primary rain forest and moist secondary growth of "bush". The genus is not considered a pest in West Africa and is in fact an economic asset, being a favourite item in the local diet of the inhabitants [79]. 1.9.3 Achatina (Achatina) achatina (LinnC) This is the name of the species of the genus and it is widely distributed throughout the forest zone of West Africa. The snail is referred to as a "giant among giants" since it is w believed to be the largest living terrestrial mollusc. It has been observed in Guinea, Sierra Leone, Liberia, the Ivory Coast, Ghana, Togo, Benin and Nigeria. It is more common to the West of the Dahomey Gap (which separates the West African forest zone into two) than to the East. Many varieties have evolved from the species and seven sub-species based on size, shape and colour have been listed [go]. The absence of geographical isolation may be due to the introduction of the eastern variety west of the Dahomey Gap or vice versa. However, since the species is more common and attains a larger size in the western part of the Gap (especially in Ghana); it is more likely that the eastern variety would have been introduced from West of the Gap. While the East African species, Achntina fitlica Bowdich, has been able to establish itself in Indonesia and on most Pacific Islands, the West African species, A. achatirza, has not been able to spread westward across the Atlantic Ocean to tropical South America. The 53 explanation for this phenomenon is not hard to find. There have been many contacts between East African and the Indo-Pacific region, but very few in the past directly between West Africa and South America, although an indirect link did exist through the slave trade from Africa via Spain or the Caribbean to South America. This lack of contact prevented the introduction of the snail to tropical South America. In recent times, there has been an increase in commercial activity between West Africa and South America, especially between the former and Brazil and Argentina. It is, however, too early to predict whether such contacts will lead to the introduction of the West African species of giant land snails to tropical South America and the Caribbean islands. Acltatina fulica has not only established itself in the Indo-Pacific region but has also become a serious agricultural pest. 'I'lie West African species of the giant land snails, however, will never assume pest proportions in West Africa so long as the human predation is so high in Ghana and other West African countries that the problem is not how to control the snails as pests, but how to conserve them and ensure that they do not face possible extinction in the future. Of the seven subspecies listed, only two are of interest, namely: Achnfina nchatina monochromatica pilsburg and Achatina nchntinn balteata Reeve: Acl~citinn nclrntina monochromatica. The subspecies monochromatica is easily differentiated from the others by having a unicolour shell, which is entirely devoid of the characteristic wavy, dark streaks associated with the species. Of more significance is the deposition of few (20-40) and relatively large (8.5 - 11.3 x 6.6 mm) eggs [92]. The deposition of large eggs might raise some doubts about its status so that anatomical studies need to be made in order to support conchological descriptions. The distribution of monochromatical is little known. It is believed to occur in Rep. of Benin and Sierra Leone, according to shells deposited in various international museums in Europe and the United States. It occurs in Rep. of Benin with certainty, but its occurrence in Sierra Leone and Guinea needs to be verified. Achatina aclzatinn balteate. This subspecies has a large, elongate-ovate to spindle- shaped cells and is much more slender than Achatina achatina. It has an uilusual interrupted distribution, which is difficult to explain. The main range is the Lower Guinea Zone from the Republic of Cameroon to Central Angola. The colonies in the Western section of the IJpper Guinea Zone (i.e. West Africa itself) are isolated and are the progeny of accidental or international introduction by man of living snails from the main Lower Guinea range [79]. 1.9.4 Common Species of the Genus Archachatina The genus Archachatina contains species that are restricted to West Africa, occurring from Sierra Leone to Zaire. Four subgenera were created [79]. The three common species occurring in West Africa belong to the subgenera calachatina. 1.9.4.1 Archachatina (Calachatina) Marginata (Swainson) This species is distributed widely, extending from Benin to Zaire, i.e. the Lower Guinea Zone. It has produced several subspecies or local varieties [79]. The shell has numerous chestnuts brown to pale brown vertical steaks, zigzag lines or blotches. The columella, outer lip and parietal wall are white or bluish white. The Dahomey gap, which separates the equatorial forest from the western forest may account for the geographical isolation of Archackati~ianzczrgi~rtlta to the east of the Gap. 1.9.4.2 Archachatina (calachatina) degneri Bequaert & Clench This species has a limited distribution compared with A. marginata. It occurs to the west of the Dahomey Gap, is a common snail in Ghana and Togo and is also found in parts of Benin. It is possible, however, that the two species, marginata and degneri, may overlap in parts of Benin, although a forest species, Archachcrtina clegneri, has invaded and successfully colonized the forest-savannah mosaic or transitional zone and may occur in wooded savannah. Unlike Achcrtinn achcrtina, Archnchatina degneri lias established itself near human habitations, occurring at such sites as refuse heaps and dumping grounds around dwellings. 1.9.4.3 A rclr uclraliira (calaclralina) veirlricosa (Gould) This species has a ventricose form (having an inflation or belly on one side) and is restricted even further west of the Dahomey Gap. It is known at present only in South-eastern Sierra Leone, Liberia (where it is one of the most common snails), and in the lower Ivory Coast 1.10 Preservation of snails The worst enemy of the giant land snails in West Africa is man. The indiscriminate gathering of the various species of Achatina and Archachntirza in West Africa may well lhreaten the species. It is, however, recognized that regulations designed to protect them may be difficult to enforce because of the popularity of the various species as a food item and the vast areas that would have to be patrolled. Attention must, therefore, be focused on their domestication. It has been demonstrated that colonies of giant West African snails can be farm-reared [84, 86, 88, 891. There are, however, numerous gaps in the knowledge of the snails and before commercial snail farms are established these gaps must be filled. The genital anatomy of three species of the giant African snails, namely: Achatina achatina, Archackatina degneri and Archachatina marginata have been studied to enhance their overlapping distribution and hybridization as well as reviewing the group classification [79Is A thorough geographical distribution and market survey of the important species of the giant land snails must be undertaken throughout the entire West African region to determine clearly their distribution and abundance. This is of particular importance in view of the conflicting reports on the distribution of the various species of the snails in West Africa. A market survey will help deternine the major collection and marketing centres throughout the year as well as the continuous price variations. The average annual earnings per family from the sale of snails at the collecting centres should also be monitored in order to assess the economic impact of the snails on the rural population. There is little information in the literature regarding the bioassays and chemical analyses of the meat and mucin from giant African snails. Snails are proteinous and also contain iron [92]. Snails are a rich source of iron and are recommended for the treatment of iron-deficiency anaemia in the tropical countries [93, 941. The chemical compositions of snails also include low methionine content but high arginine and lysine surpassing those of whole egg. 'Thus, snails may be of special value in diets low in lysine [95,96]. 5 7 1.1 1 The giant African snail: Aclratina fulica Ferussae The giant African snail (GAS) Acltatirla fulica Ferussae, has spread to nearly all countries of the tropical and sub-tropical lands. In the cause of its wanderings, it has become a great pest of gardeners and vegetable plants in all countries where it has been introduced. Much has been written about the way in which the snail was spread but many of the recent accounts prove unreliable and oAen contain inaccuracies [97]. The full story is scattered through numerous periodicals, many of them being rare and little known journals and it is felt that the true facts, in so far as they are obtainable, should be brought together. In recent years the GAS, Acltntina fulica ferussae, has greatly increased its distribution in countries of the far East. Originally a native of East Africa and Madagascar, it has become a serious pest in all countries where it has gained a footing, and the Second World War gave it a further opportunity to become established in new places in the Pacific. The snails are active nlainly during the night, resting by day in any convenient position where they are sheltered from the heat of the sun. Favourite places are crevices, under stones and decaying leaves. They also rest on trunk of trees, among the branches of hedge plants and on the walls of houses [98]. In the tropics they are most active during the rainy season and are less active at other times and even aestivate during dry weather. In Mauritius, their movements are used to forecast weather; they climb trees when rain is expected and move down again when a dry period is anticipated [99]. The length of the period of aestivation is an important factor to be considered in any comparison of rates of multiplication of the snails in different countries. All previous workers agree that Achatina is rnainly a scavenger and for this reason is more abundant in and around villages and gardens than elsewhere [98, 991. It also attacks living plants, particularly seedlings, flowering plants, vegetables, ornamental shrubs and fruit trees. Its depredations can be serious when the snail is present in many numbers. It does not do much harm to commercial plants like rubber, cocoa, tea and coconuts when established, but has been known occasionally to attack young rubber and cocoa seedlings [97]. Achatina has also been observed to drink latex from the collecting cups. Artocarpus incisa (bread fruit, bark), Artocarpus integrifolia oak, bark), Averrkoa spp. (bilimbi, flower/fruit) and Carica papaya (paw-paw, flower/fn~it),Dioscorea spp. (yam, foliage), Hibiscus escule~ztus (bark, foliage, flowers and fruits), Laportes crenulata (bark) and Solanum melongena (brinjal, bark) are attacked [99]. In captivity, the snails thrive on apples, carrots and lettuce but will eat cabbage leaves when there is nothing else available (unpublished observations on Achntina fulica var. hamillei). Mohr (1949) used slices of sweet potato, egg plant, cucumber, opuntia, etc. for feeding snails in his breeding cages.The lemon yellow, calcareous eggs are laid in batches usually buried in loose soil, in crevices and under stones or occasionally on the surface under cover of dense vegetation. The number of eggs in each batch appears to vary between 50 and 400; the egg measure 4.5 - 7.0 mm long and 4.5 mm in width. Achatina is believed to become sexr~allymature at about one year old and to continue egg laying during the remainder of its life. It is stated that individuals are not found with shells less than 60 mm in length in copula suggesting that Achatina does not reach length of 80 mm [100]. On the very conservative assumption that the sexually mature snail can live for only two reproductive seasons (i-e. two rainy seasons), it is calculated that the total number of eggs produced be about 1000 [100]. One interesting feature, self fertilization has been repeatedly observed. This means that the introduction of a single snail to a new country is enough to start a colony [I 001. Snails are haemaphrodites but they still copulate. Each has both eggs and sperms. When they mate they transfer sperm to each other - cross fertilization. The incubation of the eggs lasts from one to 10 days. Observations on the African form, fulica var. hamillei indicate that the larvae take one or many days to hatch and that they may remain below the surface of the soil for 5 - 15 days before coming to the surface [loll. 1.12 Physical requirement and production Becai~seof our relatively mild climate throughout the tropics, snail production can be largely done outside. In some of the colder European countries where snail production is a much bigger industry, producers have been forced to develop indoor techniques of production, which creates extra problem 1641. There are a number of key essential requirements: 1. Soil -- Snails need access to soil to reproduce and grow in a healthy manner. Research has been shown that the soil provides the necessary nutrients for them. Ideally, the soil pH levels should be in the 5 - G range. The soil should not be too hard because they oviposit (lay eggs) under the soil's surface. 2. Nutrition - A good regular supply of nutrients, especially calcium, is essential. They eat a variety of foods, especially soft vegetable material (leaves), fruits and even left- overs from the kitchen. 3. Moisture -- Conditions must be kept moist to encourage production and growth. However, it should not be waterlogged because snails are susceptible to very low humidity and dry atmosphere. 4. Density - Snails are susceptible to overcrowding. The recommended levels vary but it seems that when they are breeding, you should only have 6 - 8 snails/square metre. Young, smaller snails can be run at much higher densities. 5. Shelter - They are susceptible to extremes in temperature and wind. The usual production unit is a shade cloth enclosure with plenty of vegetation or artificial shelter (over turned boxes etc.) to allow the snails to get away from the heat, the cold or the wind. Temperature is important. They appear to like conditions of about 20 " C with high humidity. If the temperature gets above 28 ' C or below 5 " C then they tend to cease activity. So the physical requirements for snail production are quite cheap and simple but attention must be given to ensuring that the "climate" and soil in the shade house is optimum for the snails WI. Nutrition is important. Some producers use scrap vegetables, which are ideal but the older scraps must be removed before they start to rot, othenvise it will affect the snail. Therefore, this can be a labour intensive way of doing it. Other producers use bran based foods, which must be kept d~y.Snails require high protein and high fibre foods with plenty of calciun~(Ca). This is essential for shell development. Also, they require trace elements, ash and vitamins A, D, B12and E [64]. 1.13 The control of Achatina fulica Various methods are employed to keep down the numbers of Achatina in infested areas; there is no certain cure and all that can be done at present is to keep the snail population within reasonable proportions. Positive control measures, such as hand picking and the use of chemicals as well as direct means involving protection of plants and biological control: 1.lXl Chemical control (a) Poisoned Lime wash: Like all gastropods with a thick shell, AchatinaBtlica must have lime to consolidate its shell. In countries where there is a lime deficiency in the soil, Achntiim scrapes off the lime from white-washed walls, mortar and cement and will even enter houses in search of it. Such walls, sprayed with a 1 % solution of calcium arsenate Ca3 can be used as a lure for killing off the snails. The latter however take several days to die and may wander over a considerable distance during that time. Consequently, few of the dead snails can be picked up and buried; the dangers of having such putrifying snails lying around human habitations have been pointed out [102]. (b) Use of copper sulphate [98] and (c) The use of meta-bran baits [103]. 1.13.2 Collection and destruction (a) Hand-picking: - Hand-picking of eggs and shells of ail seizes seem the only satisfactory way of keeping down the Achatina population. As Achatina is largely nocturnal, the best time for collecting are very early in the morning and late in the evening, but where there is a plague a good percentage can be picked up in the day time. Collecting by hand can be assisted by the preparation of suitable traps made of rubbish, bricks or stones in suitable places. The snails will take refuge under any cover provided. For these traps to be effective they must be inspected daily and all snails destroyed [97]. (b) Crushing: - Achatina is cannibalistic, but only when it finds a maimed or crushed individual, and some use has been made of this characteristic to lure the snail to its destruction [97]. (c) Destruction is another method of effectively eliminating snails [98]. 1 .l3.3 Other con trot and protective measures There are many other options in controlling snails like destruction of garbage and village refuse [I 041, clean weeding [105], protection of small gardens and fruit trees [98]. 1.1 3.4 Biological control It is well known that Achatina is no pest in its African home and it is generally assumed that it has natural enemies, which effectively control its numbers. Unfortunately, little is known of these [97]. The most valuable enemy of Achatina undoubtedly is the larvae of the Indian glow-worm, Lamp-oph-yrus tenebrosus [106] in Ceylon, also pond tortoise of Ceylon [99]. Also, soldier ants are enemies to snails and it is difficult to extricate them from snails because when they sting the snail it withdraws into its shell along with the ant. 1.14 In search of protein: snailery As the prices of beef and fish soar beyond the average citizen's reach, Nigerians will have to turn to other sources of protein. But if only they are aware these sources are readily available in their environment: shell-fish - snails, periwinkles, clams, oysters, octopuses, scallops, squids - are a feature of Nigerian's natural environment. They are, in fact, so common that people take them for granted. Yet, they constitute a major source of proteins, which Nigerians are finding it harder to come by everyday following beef and fish shortage. And who would imagine that insects - some of which man have come to associate with destruction --- are in fact, a repository of human protein needs. Snails protein content compares favourably with that of beef, but they are low on fats, unlike beef. However, they have a special substance galacton - a special fonn of carbohydrate found in their abdominal glands. The galacton also has immi~nologicalvalue, about which research is being stepped up in Germany [ 1071. 1.15 Analgesic potentials of snails Scientists may have stumbled upon a compound in snails, which could form the basis of a new class of medication to ease clvonic pain. But alternative medicine practitioners in the country reported that the finding is a reinforcenlent of their contention that snail generally holds a promise for treatment of pain [108]. Indeed, the team of scientists from the University of Melbourne, Australia reported that a compound found in a toxic marine snail could offer chronic pain sufferers some relief. They were so confident of the toxin's potential to treat long- term pain associated with disease such as cancer, ADS and arthritis; they have taken out of a patent on it. They were also seeking a commercial partner to take the substance to human trials. Essentially, the compound comes from a species of cone shell, a potentially deadly type of mollusc found on the Great Barrier Reef. In the laboratory studies so far, the drug, known as ACVI, is said to have proved more powerful and longer lasting than morphine. Researchers say the drug does not have the same side effects, namely: constipation and respiratory problems. Also, unlike morphine it is not addictive [108]. ACVI is said to block the transmission of pain along the peripheral nervous system -- the system that runs throughout the body and transmits the pain people feel. 1.16 Objectives of the study This research study was aimed at: - Extraction and characterization of mucin from African giant snail. - Utilization of the characterized snail mucin in ihe formulation of bioadhesive chlopropamide granules CHAPTER TWO MATERIALS AND METHODS 2.1 Materials 'T'he following materials were procured from their local suppliers and used without further purification: potassium chloride, copper sulphate, tragacanth powder, sodium chloride, monobasic potassium phosphate, calcium chloride, charcoal meal, Polysorbate (Tween 85), safranin red, Gentian violet, malachite green and aluminium oxide (Merck); gelatin, sodium carboxymetliylcell~~lose(SCMC) (Aqualon Co. USA), blue dextran, methyl red, bovine serum albumin (BSA) and ovalbumin (Sigma, USA), Sephadex G-200 (Pharmacia, England), Carbopol-940TM and Carbopol UltrezTM 10 polymer (B.F. Goodrich, USA); ethanol, hydrochloric acid, copper sulphate, conc. nitric acid, sodium acetate, silver nitrate sodium hydroxide, tetraoxosulphate (vi) acid, ammonia methyl alcohol, Ethylene-diamine-tetraacetic acid (EDTA), and I-naphthol (BDH, England). Simulated gastric fluid (SGF) and simulated intestinal fluid (SF) were prepared following the compendium (USP XXIII) [log] specification. Distilled water was obtained from a batch in the Pharmaceutics Laboratoiy. All other reagents and solvents were of analytical grade and were used as such. 2.1.1 Snails The giant African land snails (Archachatina marginata) used were procured from Ibagwa-Nkwo market in Nsukka zone of Enugu State. A total of 450 snails were used. 2.1.2 Animals The various experimental animals used were: Cavia porcellus (Guinea pig), Mus musculus (mice), Rattus norvegicus; strain: Sprague-Dawley, (rat) and Large white pig (Porcine spp.). 'The weights of the animals were measured with an animal balance (W.B. Nicolson St. Ltd, CAasgow). 2.2 Methods 2.2.1 Extraction of snail mucin (slime) Afler procurement, the shells of the giant African land snails were knocked open at the apex and a spirally coiled rod inserted to remove the fleshy body from which the excretory parts were ren~oved.The fleshy parts were then placed in 250 ml of water and washed. Further washing with 250 ml portions were carried out for three times. These washings were pooled together in a plastic bucket and lyophilized in a lyophilizer (Baird & Tatlock, Q. No. MF 45, England.) . The greyish-brown lyophilized flakes of the snail mucin were pulverized into fine powder using a mortar and pestle and stored in an air-tight container until used. 2.2.2 Pliysicoclie~nicaltests Preliminary physicochemical tests were carried out on the lyophilized powder. 2.2.2.1 Test for sugars Freshly prepared Fehlings solutions A and B (few drops) were added to 1 ml of 1 % w/v aqueous dispersion of snail mucin and then heated in a boiling water bath for 5 min and observed. 2.2.2.2 Test for carbohydrates (reduction test) a. To 1 nd of the snail mucin (1 %w/v) dispersion was added 1 % iodine solution (few drops) and then observed for blue-black colouration. To about 0.1 g of the powdered extract was added 2 drops of iodine solution and observed. b. Moliscb's test This reaction is a general test for the presence of carbohydrate and other organic compounds that could form furfuraldehyde (furfural) or hydroxymethyl furfuraldehyde (hydroxy-methylfurfi~ral)in the presence of concentrated sulphuric acid. In the Molisch's test, the basic principle is one in which concentrated sulphuric acid hydrolyses glycosidic bonds to give the monosaccharides, which are then dehydrated to furfural and its derivatives. For the test, two drops of a-naphthol solution was added to 2 ml of the snail mucin dispersion and mixed thoroughly. Then 1 rnl quantity of concentrated sulphuric acid was carefully poured down the side of the tube and observed. c. Tollee's reagent test The Ag-t ions in a solution containing silver ammonia complex are reduced to metallic Ag by aldehydes, reducing sugars, polyhydroxyphenols, aminophenols, formic acid and other reducing agents. Tollen's reagent prepared as 1 in1 of 5 % silver nitrate solution was treated with a few drops of 5 % sodium hydroxide solution. A volume of aqueous ammonia just enough to redissolve the precipitate was added to 3 drops of the snail mucin dispersion and the mixture warmed in a boiling water bath for a few minutes. The colour of the precipitate formed was observed. 2.2.2.3 Test for Proteins (amines; oxidation test) a. Bitwet test To about 10 mg of snail mucin powder in a test tube, a few drops of water and 1 ml of dilute sodium hydroxide were added. A 1 % copper sulphate solution was added dropwisely and the solution shaken thoroughly after each drop and observed. b. Milloris test A 5 ml quantity of Millons reagent was added to a 2 ml of the extract dispersion in a test tube, heated for 5 min and observed. c Xanthoproteic reaction To a dispersion of the snail mucin, a few drops of concentrated nitric acid were carefully added. A white precipitate was formed, which turns yellow on heating. The contents of the test tube were cooled and a few drops of ammonia solution added and precipitate observed. 2.2.2.4 Test for fixed oils A drop of the acetone extract was placed on a filter paper. The solvent was allowed to evaporate and the filter paper observed carefully. 2.2.2.5 Solt~bilityprofile of the mucin 'The solubility of snail much in several solvents was determined by dispersing definite quantities of the slime in a definite volume of each solvent -water, acetone, ethanol, 0.1 M solution each of sodium hydroxide, ammonium hydroxide, sulphuric acid and hydrochloric acid and dimethylsulplioxide (DMSO) at different temperatures (27 OC, 35 "C, 40 OC) and the solubility in each solvent was recorded. 2.2.3. Preparation of snail mwin dispersion The respective quantities of 1 g, 2 g, 3 g, 4 g and 5 g of powdered snail mucin extract were weighed using the sauter balance (August Sauter, KG-D 7470 Germany) and each dispersed in 25 ml of water contained in a volumetric flask. These dispersions were stirred for 15 mins and allowed to hydrate completely for 24 hours before use. Thus, the various concentrations of the snail mucin were 4, 8, 12, I6 and 20 % wlv respectively. 2.2.4 Rheological study on aqueous snail rnucin dispersion The rheology of the much (20 % wlv) was studied with viscometer (Haake Rotovisco Model RVl, NJ 07662) using concentric cylinder assembly. The viscosity was determined at different shear rates designated by the fixed speeds of the viscometer. he viscosity was plotted against the speeds of the viscometer. This determination was at 30 "C. 2.2.5 Type of flow The nature of flow of the mucin dispersion (20 ?h wlv) was determined at 30 OC. The shearing stress obtained from the scale of the viscometer was plotted against the rate of shear. 2.2.6 Effect of concentration of the mucin Dispersions containing 4, 8, 12, 16 and 20 % wlv of the mucin were prepared and their viscosities determined with the viscometer at 30 "C. 2.2.7 Effect of temperature The viscosity of the 20 O/u w/v snail mucin sample was determined at 32 O, 34 ', 36 O, 38 ", 40 O, 42 ", 46 ", 48 ", 50 ", 60 O and 70 "C with the viscometer set at gear 9 and the effect on the viscosity of the n~ucindetermined. 2.2.8 Effect of electrolytes The electrolytes used were potassiuni chloride, copper sulphate and aluminium oxide. Calculated anio~intsof 0.2, 0.5, 1 .O, 1.5 and 2.0 g of the various electrolytes were dissolved in 20 % wlv dispersion of the snail tnucin respectively. The viscosity of each sample was determined with the viscometer at 30 "C. 2.2.9 Effect of polymers The polymers used were gelatin, sodium carboxyrnetliylcellulose (SCMC) and Carbomer-940. Calculated amounts of 1 : 1, 1:2, 1 :3, 1 :4, and 1:5 of the various polymers were each mixed with snail mucin and dissolved in enough distilled water to yield a 20 % w/v dispersion of snail much respectively. The viscosity of each sample was determined with the viscometer. 2.2.10 Effect of aging Dispersions of snail much in distilled water were prepared to contain 4, 8, 12, 16 and 20 O/u wlv of the snail mucin. The viscosity of all the sanlples was determined at 30 'C and thereafter, weekly for a period of 4 weeks. The effect of aging was also determined on the solid mucin 2.2.11 Molecular weight determination by gel permeation chromatography on cross- linked dextran G-200 The cross-linked dextran G-200 was allowed to swell in excess buffer (0.05 M Tris buffer, pH 7.5) for the recommended time of three days at room temperature, in order to obtain satisfactory flow rates through the gel and good separation. The column was poured and equilibrated with the buffer. The void volume was established with blue dextran (V,) (5 mgtnil; weight average-molecular weight 2 x 10" read at 625 nm). The sample (snail much dispersion) was applied. The volume at which the active fraction snail mucin eluted from the column was determined (V,). Four standards (I 0 mg/ml) were applied to the column in runs of lwo standards per run to determine the elution volumes (V,) of the standards. The standards used were methyl red, bovine serum albumin (BSA).;ribonuclease and ovalbumin. The Kav for the active fractio~i(snail mucin) and the standards were calculated using Eqn. 13 Eqn. 13 where V, is elution volume of the (active) material, V, is elution volume of blue dextran, V, is {hetotal volume of gel bed (sn r 2h), r is radius of the column and h is height of the column. To prepare the standard curve, K,, of the standards was plotted against log molecular weight. From the Kavof the active material (snail much), which was unknown, the molecular weight was determined from the standard curve. 2.2.12 Determination of snail rnucin isoelectric point Seven buffer solutions of different pH values ranging over 3.2 - 5.7 were made in 7 test tubes (Table I). A 0.5 ml volume of 2 % wlv snail mucin (protein solution) was added to each test tube and the contents mixed. The test tubes were shaken and noted for the appearance of a Table 1. Br~ffercomposition and solution pH. Test tube number Buffer mixture composition (ml) Solution cloudy solution. A 2 ml quantity of ethanol was added to each of these test tubes and visual estimation of the degree of cloudiness was determined. 2.2.13 'Test for allergic properties 2.2.13.1 Preparation of stock solrrtion A 4 % w/v stock aqueous dispersion of snail mucin was prepared. 2.2.13.2 Crrinea pig wheal test (Local anaesthetic property) A total of four guinea pigs were used due to seasonal scarcity of guinea pigs. The guinea pigs were prepared 24 h before the test by first dipping and then shaving the hair on the lower back. The skin sensitivity was greatest in the midline and slightly more in the upper part than in the lower part. A sterile sharp 26 G x inch needle was used for each injection. The skin on the lower back of the guinea pig was stretched taut by holding the animal with the hand placed around the abdomen and by pulling the skin taut with the thumb and forefinger. The mucin was injected in the same direction as that in which the skin was being held and the needle inserted into the dermis or epidermis. When the plunger of the syringe was withdrawn there was no appearance of blood indicating an intradermal injection. The volume injected (5 ml ) was enough to raise a wheal, which was outlined. Five min after the injection, sensitivity of the area was tested by pricking the skin at the injection site with a needle, six times lightly, and, as a control, the skin as far away from the injected site as possible. Six twitches was recorded for this control test, but the responses at the site of the injection will indicate the degree of anaesthesia, which is expressed as the number of negative responses i.e. of failure to twitch; 616 indicates maximum anaesthesia and 016 indicates no anaesthesia. The test was repeated at 5 min intervals for a period of 30 min after the injection. The total score for each wheal was added up and expressed as the total number of negative responses out of 36 possible. The above procedure was repeated for the other doses of the stock solution of the snail mucin. 2.2.14 'I'oxicological studies 2.2.14.1 LDSodetermillation (Acnte toxicity study) This was done using Lorke's method [110]. A total of 24 Swiss albino mice were fasted over night although with access to water. They were divided into four groups of six each and their average body weights determined to be 20 g. The snail mucin was prepared as a suspension in tragacanth solution to help uniformly suspend the dispersed particles. A 1.0 g quantity of tragacanth powder was dispersed in 50 ml of water to obtain a 2 % stock solution. Three groups of six mice were given different doses of the snail mucin-tragacanth combination in the order of 1 .O, 3.0 and 5.0 glkg body weight of the mice while the fourth group (control) received 200 mglkg tragacanth placebo (Table 2). The number of mice that died in each group within 12 h from the time of administration was noted. 2.2.14.2 Chronic toxicity study i. IIaematology The study comprised of 24 Sprague-Dawley albino male rats of ten weeks old. The 24 rats were weighed individually on the first day and their weight range gave 85-95 g. They were put into four separate cages such that each cage contained six rats. They were labelled using safranin red for group A, gentian violet for group B, malachite green for group C and charcoal for group D (control). Their blood samples were coIIected through the eye vein, using capillary tubes and dropped into labelled EDTA-contained bottles with tight closures and shaken well to prevent clot formation. The blood samples were analysed for packed cell volume (PCV), Table 2. Dosing pattern for LDSodetermination Av. body weight Mucin doses administered of mice (g) orally (gkg) b.w. Snail ~nucirl Snail niucin Snail mucin Tragacanth placebo erythrocyte sedimentation rate (ESR), total leucocytes count (TLC) and differential leucocyte count (DLC) before dosing with the snail mucin. The snail mucin was prepared as a suspension in 2 % tragacanth solution to uniformly suspend the dispersed particles. The rats were dosed in such a way that group A was given 750 mgkg, group B 1,500 mglkg and group C 3,000 mglkg. Group D rather was given the tragacanth placebo 500 mglkg (Table 3). This was repeated daily for twenty-one (21) days. At the end of the twenty-first day, the blood samples of the rats were withdrawn again and analysed as before. ii. Histopatl~ology The rats were then sacrificed and their livers and kidneys removed and preserved in labelled formalill bottles. 'These tissues were specially treated and fixed unto slides, which in turn were viewed under the microscope and snapped using a special camera (Microphotograph Machine Leica Galen 111, Leica Inc. USA). The films were developed to study the effect of the snail mucin chronically on the tissues of the liver and kidneys and probably determine to what extent this could be harmful to the named tissues. 2.2.15 'I'ensiornetric determination of bioadliesive strength of snail much 2.2.15.1 Preparation of snail mrrcin stock The respective quantities of 1 g, 2 g, 3 g, 4 g and 5 g of powdered snail mucin were weighed using a weighing balance (August Sauter, KG-D 7470, Germany) and each dispersed in 25 nil of water contained in volumetric flasks. These dispersions were stirred for 15 min and Table 3. Dosing pattern of the snail mucin for the chronic toxicity test ------Drug Dose (mglkg) Rat groups Snail ~nucin 750 A Snail mucin 1500 B Snail mucin 3000 C Tragacanth placebo 500 D ------allowed to hydrate completely for 24 11 to give 4, 8, 12, 16 and 20 % w/v dispersions respectively. 2.2.15.2 Tellsiometric bioadhesive test A tensiometer (Lecomte Du Nuoy Tensiometer, Model Nr 3124, A. Kruss Germany) was used for this study. A sniooth polythene support was secured on a platforn~(a component of the tensiometer) with an adhesive. A freshly excised pig jejunum was thoroughly washed of its waste material. A sniall portion of the pig jejunum with diameter 2 cm and length 5 cm was used. The mucus surface of the intestines was exposed and used immediately for the test. The tissue was pinned unto tlie polythene support of the bioadhesive instrument placed on a metal support. The instrument was zeroed and the bioadhesion of the clean glass plate determined in degrees by gradually raising the platform such that the plate on the lever arm of the tensiometer was in contact with the tissue and 7 niin contact time allowed for interaction to take place. The glass plate was raised by means of screw until it just detached from the surface of the tissue. The force required to remove the glass plate froni the surface of the tissue was read off from the microform balance in degrees. The glass plate was washed, dried and subsequently coated to a thickness of 2 mm with the different aqueous dispersions of tlie snail mucin concentrations and allowed to dry for 15 min. This was rcpeated six times for each concentration of the snail much aqueous dispersion and the forces required to detach the glass plate from the tissue were recorded in degrees and conversions of these to tension were done using Eq. 14 [7,11l-1171. ...... Eqn. 14 79 where T is tension equivalent to the bioadhesive strength, M is the mass required to return the lever to zero position after each bioadhesive experiment, L is the area of the bioadhesive interface, F is the instrument constant (0.94) and g is acceleration due to gravity. 2.2.16 Evaluation of tlie bioadhesive strength of the snail mucin using coated glass beads 2.2.16.1 Preparation of simulated gastric fluid (SGP) without pepsin A 2.0 g quantity of sodium chloride was dissolved in sufficient distilled water and 7.0 ml of hydrochloric acid added, and the solution was made up to 1000 ml with distilled water. The pH of tlie solution was adjusted to 1.2 using a pH meter (model 290 MK, Pye Unicam). 2.2.16.2 Preparation of simulated intesti~~alfluid (SIR without pancreatin A 6.8 g quantity of monobasic potassium phosphate (K2HP04)was dissolved in 250 ml of water and 190 ml of 0.2 N sodium hydroxide (NaOH) and 400 ml of water were added. The resulting solution was adjusted to a pH of 7.5 * 0.1 with 0.2 N NaOH, and later diluted with water to 1000 ml. 2.2.16.3 Coating of glass beads Glass beads of diameter 3.0 mm and average weight 56 mg were cleansed with water and acetone thoroughly to maximize the roughness factor. They were allowed to dry. Different concentrations of the snail muciri dispersions prepared as in section 2.2.15.1 were used to coat the glass beads to an average weight of 65 mg, by successive dipping in the mucin dispersion, air drying and storage in a desiccator until used. 2.2.1 6.4 'The bioadhesion test: rise of coated glass beads The apparatus designed and used in this study consisted of a separating funnel clamped to a retort stand with a rubber tube attached at the end of the funnel (Scheme 2). A metal support was used to position a plastic support at an angle of 30 O. Freshly excised pig jejunum (1.7 x 15.0 cm) was pinned on the plastic support, and a beaker was placed directly under the plastic support to collect the detached beads and the detaching solvent. Fifteen coated glass beads were placed on the exposed much surface of the tissue. Mucus-polymer interaction and polyner hydration was allowed to take place over a period of 7 mill. Simulated gastric fluid (SGF) without pepsin (250 ml, pH 1.2) contained in separating funnels, was allowed to flow over the coated glass beads at a rate of 30 ml per min. The number of undetached beads were noted and used as a measure of bioadhesion. The experiment was repeated five times for each concentration of the polymer and the average value recorded and analysed statistically. This procedure was repeated for simulated intestinal fluid (SIF) without pancreatin (250 ml, pH 7.5 * 0.1). 2.2.17 Rioadl~esio~~of snail ~nucingranules 2.2.17.1 Preparation of granules Different batches of chlorpropamide granules were prepared to contain 1 :1, 1:2, 2: 1, 0: 1 and 1:O combinations of Carbopol ultrez-LOTMand snail mucin with chlorpropamide. The granules were prepared by wet granulation, as in tablet production. The dried granules were sized and those falling within a size range of 1 .- 2 mm were used for the bioadhesion study. Scheme 2: Set-up for bioadhesion test 82 2.2.17.2 Bioadhesion test on the granl~les The apparatus designed for the coated bead experiment above was used. In this instance however, a 1 g quantity of the granules was uniformly spread on the everted tissue. At the end of the SGF flow the undetached granules were recovered, dried and weighed. The bioadhesion percent was evaluated. This was repeated five times for all the batches. 2.2.18 Determination of the granule micromeritics 2.2.18.1 Flow rate and angle of repose The funnel niethod described by Carstensen and Chan [ 1 181 was employed to measure the flow rate. A weighed quantity of the snail mucin was introduced into a plastic funnel with the following dimensions: - efflux tube length G cm. - diameter of funnel measured from bottom of efflux tube 8 cm. - diameter of funnel at top 6.2 cm. - diameter of efflux tube 0.80 cm. A 10 g quantity of the granule was allowed to fall freely onto a weighed piece of paper whose area had also been determined. The time of flow was noted. The resulting heap height, 11, was measured with a meter rule. The diameter, d, of the heap base was also measured. The flow rate, F, was computed from Eqn. 15. F= Mass(g) ...... Eqn. 15 Flow time(s) The angle of repose, 8, was calculated from Eqn. 16. Average of five determinations was taken. 2.2.18.2 Bulk and tapped densities A 10 g quantity of granules were weighed and gently introduced into a clean, dry 50 cc measuring cylinder calibrated in cm! The volume of the granules was read without tapping. The bulk density (Dl,) was determined from the bulk volume by the following relationship: Mass Db (~~rn-3)...... Eqn.17 - Flow Volume The tapped density (D,) was determined by uniformly tapping the cylinder on a flat firm surface until there was no change in volume. Mass ~1~= (~~rn-') ...... Eqn.18 Flow volume An average of three determinations was taken. Hausner's quotient (HQ) and percentage coinpressibility (PC) were calculated using the followiiig equations: PC=- vb-vtxlOO ...... Eqn. 20 Vb where V, is the tapped volume, Vb is bulk volume. 2.2.19 Gastrointestinal motility test in mice Fifteen albino mice of either sex (20 - 25 g) were randomly divided into five groups of 3 animals per group. The animals were fasted for 24 h prior to the experiment, but had free 84 access to water. One group received polysorbate [Tween 85TM(20 mlkg)], the second group received atropine (10 mglkg) while the remaining 3 groups received different doses of the snail mucin, (100 - 400 mpjkg). All administrations were by the oral route. Five minutes after drug administration, 0.5 ml of a 5 % charcoal suspension in 3 % aqueous solution of Tween 85 was administered to each animal orally. The animals were sacrificed 30 mins later and the abdomen opened. The percentage distance travelled by the charcoal plug in the small intestine (from the pylorus to the caecum) in the treatment groups was determined [I 19, 1201. 2.2.20 Deterriiination of hrnax of absorption A 1.0 1ng quantity of pure chlorpropalnide powder was weighed using a balance. This was dissolved in 100 ml of simulated intestinal fluid (SF, pH 7.5 * 0.1) and scanned using a spectropl~oton~cter(UNICO-UV 2010 PC) at the range of 200-400 nni to produce a spectrum (Fig. 1). 'The maximum wavelength of absorption (hmax) was noted. 2.2.21 Calibration curve of clilorpropamide in SIF (Beer's plot) Increasing concentrations of chlorpropamide was prepared in SIF to contain 0.01, 0.02, 0.03, 0.04 ad0.05 nlg %. Their absorbances were read off in a spectrophotometer (UNICO- UV 2010 PC) at '229.7 nm using SLF as the blank. The absorbances were plotted against the concentrations of the solutions to get the Beer's plot. 2.2.22 Absolate drug content For each granule batch, 0.5 g of the formulated granule was weighed in a balance and placed differently in a 100 ml volumetric flask. A 70 ml volume of SF (pH 7.5 * 0.1) was poured into each of these volumetric flasks and the granules allowed to hydrate for 24 h. The Test Report Test Date: 09-23-2003 User Name: Petra 'Test Mode: SCANNING Gra~h'sName: Uv scan of chlorpropamide in SIF Start Wavelength: 200.0 nm End Wavelength: 400.0 nm Scan Interval : O.lnm nm Abs peak: Fig. 1. UV Scan of chlorpropamide in SIF 86 contents were thereaAer filtered and 0.25 ml of each filtrate made up to 25 ml with the SIF. The absorbances were read off using a spectrophotometer (UNICO-UV 2010 PC) at 229.7 nm. This was repeated five times and was done for all the batches. The drug contents were calculated with reference to the Beer's plot for chlorpropamide in SF. 2.2.23 Release studies The release of the drug was assessed in a magnetic stirrer hot plate assembly (Model SR. No. IUM 52188, Remi Equipment, India) using 500 ml of SIF (pH 7.5 * 0.1) as the dissolution medium [I 141. The medium was maintained at 37 * 1.0 OC throughout the test period. At zero time, 0.5 g of the formulated granule was put inside the dissolution medium. At predetermined times of 5, 15, 30, 45, 60, 90, 120 and 180 min, 5 ml of the dissolution medium was withdrawn, adequately diluted, assayed spectrophotometrically. Each amount of dissolution medium withdrawn was immediately replaced with equivalent amount of fresh dissolution medium. The concentration of chlorpropamide released during each period was determined with reference to Beer's plot in SIF. The data generated was further analysed graphically. The tso and the tzo, which are the time for 50% and 20 % of the drug to be released, were extrapolated from the drug release profiles. The release was further analysed using the Higuchi's square root model [12 11 as well as the Fick's model [I221 for the amount of drug CHAPTER THREE RESULTS AND DISCUSSION 3.1 Physicochemical properties of snail mucin Results of some physicochemical tests performed on the snail mucin showed that carbohydrate, sugar, proteins and traces of fats were present as presented in Table 4. In both hydrated and dry states, the mucin is light brownish in colour, almost tasteless and has a characteristic pleasant odour. It was found that snail mucin when dispersed in water gives a slight viscous dispersion. This is unlike gelatin - a typical purified animal protein that swells in cold water and gives a colloidal solution when heated forming a more or less firm gel. In cold water, snail mucin disperses with a little difficulty. The snail mucin is not soluble in all the organic solvents tested except DMSO (Table 5). It is also not soluble in O.1M (NaOH, HCI, &So4 ,NH40H), the temperature of dissolution notwithstanding. 3.2 Rlleological test on aqueous dispersion of snail mucin The intended utilization of snail mucin in pharmaceutical preparations necessitated the study of the effects of various factors on the viscosity of the mucin. The flow properties of all . . '- . . .,..- 41- - .,,-:r'....v:4-..,,,A Table 4. Physicocl~emicalproperties of snail mucin Test Observation Inference ------.-- - - Simple sugar +-I-+ Present Carbohydrates +-ti- Present Proteins +ft Present Fats + 'Trace amount + Presetit in trace amount; ft+copiously present Table 5. Solubility of snail much in some common solvents Acetone ROII 0.1 M 0.1 M 0.1 M 0.1 M DMSO NaOH HzS04 NH40H HCI - Quite insoluble + slightly soluble 'The viscosity of the mucin was determined with the help of viscometer (Haake Rotovisco Inc. rnodel RVI, NJ 07662). But for the study of rheology, a sample of the fresh 20 % wlv dispersion of the snail much was subjected to progressively increasing shear rate. The scale reading at each of the fixed shear rate (speed) was uoted and the shearing stress was obtained by multiplying the scale reading with the relevant instrument constant in accordance with Eqn. 21 11 = IJSK ...... , . . . Eqn. 21 wherc q is the viscosity, K is the sensor constant 0.261, S is the instrument reading and U is the speed factor equivalent to 64.8. The result of the relationship between speed and viscosity of the 20 % w/v dispersion is presented in Fig. 2. This shows that viscosity has a linear relationship with speed. 3.2.1 Type of flow The result of the flow property of the snail mucin studied using fresh 2 % wlv dispersion is presented in Fig. 3. The result indicates that snail mucin demonstrated a pseudoplastic type of flow since at higher shear stresses, the flow curve tended towards linearity indicating that a minitnun1 viscosity has been attained. This is probably due to the presence of long high molecular weight nlolecules in solution resulting in entanglement alongside the association of immobilized solvent so that under the influence of shear, the tnolecules disentangle and align themselves in the o! 1 1 1 1 I d 012345678910 Speed Fig. 2. The relationship between speed and dscosity using a 20 %wlv dispersion of snail rnucin 0 2 4 6 8 10 Shearing stress I I 1 0 5 10 15 20 Concentration (% wlv) Fig. 4. Effect of concentration on viscosity of the snail much dispersions 3.2.3 Effect of temperature Fig. 5 shows the effect of temperature on the snail nlucin dispersion. Increase in the temperature of 20 % w/v dispersion of the snail mucin rapidly decreased the viscosity (Fig. 5). The viscosity of the rnucin was determined with the viscometer set at speed 9. The rupture of the sub-units held together by inter-chain disulyhide bonds and some non-covalent interactions 1591 between the mucus glycoproteins by heat is probably responsible for the decrease in viscosity, the end result being a loss of the visco-elastic characteristic of this mucus secretion. 3.2.4 Effect of electrolyte Fig.6 shows the result of the effect of electrolytes on the viscosity of snail mucin dispersion. The figure shows that the electrolytes decreased the viscosity of the mucin dispersion depending on the hydrolysis and precipitation of the nlucin by the electrolyte. However, the trivalent electrolyte (A1203)had the highest reduction of viscosity probably due to its valency, which characterizes its precipitating power. This was followed by the divalent (CuS04) and finally by the univalent electrolyte (KCI). This suggests that snail mucin is anionic and electrolytes reduce the viscosity of their system thus high concentrations of the mucin have to be employed in vehicles where ionizable drugs are present. 40 50 60 70 Temperature (oC) Fig. 5. Effect of temperature on the viscosity of 20 %wh dispersion of snail mucin in distilled water 0 1 2 3 4 5 6 Concentration (%wlv) Fig. 6. Effect of elctrolyte on the viscosity of 20 %wlv dispersion of snail mucin Without electrolyte, KCI, A CuS04,AAI2O3 1 2 3 4 5 Concentration (%wlv) Fig. 7. Effect of polymers on the viscosity of 20 %w/v dispersion of snail much Without polymer mGeiatin Bl SCMC BCarbopol940 Carbopol-940 whose aqneous solution is acidic also demonstrated an increase in the viscosity of the snail nlucin dispersion, The increased viscosity produced between Carbopol 940 and snail mucin was higher than other polymers. There may have been greater entanglements in this combination than in other plyrners leading to rheological synergism. Since these polymers - gelatin, SCMC and Carbopol 940 enhanced the viscosity of the snail mucin dispersion, they can be employed as viscosity enhancers together with snail mucin in suspensions, tablet coating, binders or disintegrants in dosage fornlulations. 3.2.6 Effect of aging Ikcrease in the viscosity of snail mucin dispersion was observed (Fig. 8) with time. This may probably be due to the hydrolysis of the nlucin in the aqueous medium and to the decomposition of the mucin by some enzymes. This could be hazardous in emulsion by causing thinning. However, the solid mucin was stable. 3.3 Molecular weight determination of the snail much by gel permeation cllrom~tographyon cross-linked dextran G-200 Proteins of known ~nolecular weight - methyl red, bovine serum albumin (RSA), ribonuclease and ovalbumin - were chosen as reference proteins and their elution volumes measured for each of the columns used. In addition, the void volume (V,) of each column (elution volunie of substances completely excluded from the gel pores) was measured in experiment with blue dextran plus reference proteins. The molecular weight of the snail mucin was estimated from the calibration curve of the standards (Fig. 9) to be 4, 281 Daltons. The useful working range of cross-linked dextran G-200 depends on the extent to which the gel has swollen, and evidently varies also from lot to lot [123]. The lower molecular-weight limit for useful fractionation of polypeptides and proteins is approximately 5000 Daltons, whereas the upper limit may be within 500,000 to 1,000,000 Daltons or even higher, depending on the gel. The lower molecular weight limit for complete exclusion fiom the pores of cross-linked dextran G-200 will also depend on the gel. 0 10 20 30 Time (days) Fig. 8. Effect of aging on the viscosity of snail mucin dispersions 2 4 8 Log. Mol. Wt. Fig. 9. Plot of Kav versus log molecular weight { the Kav values include: Methyl red (2.473); Snail much (1.351); Ovalbumin (1 .I16); BSA (1.026)) 3.4 Isoelectric point (IEP) determination of the snail mucin Protein as an amplioteric polyelectrolyte carries both positive and negative charges whose ratio is defined by the number of acidic and basic amino acids in the protein molecule. The charge on a protein molecule is a factor of protein stability in solution, since it prevents the agglomeration of protein particles and their precipitation. The net charge of a protein macronioleculeis affected by the medium pH. Each protein is characterized by a pH value at which the sum of positive and negative charges on the protein is equal to zero. This state of protein is referrcd to as isoelectric, and the corresponding pH value is referred to as isoelectric point (I.P.). At I.P., the protein solutions are unstable, and the proteins are prone to easily deposit as a precipitate, especially in the presence of dehydrating agents (ethanol, acetone and others). This is probably due to the presence of zwitterions in the medium so that the protein molecule (snail mucin) is least solube, resulting in cloudiness whose degree was most at the pH of 3. 4. Therefore, the isoelectric point of the snail mucin was found to be 3.4. Below the isoelectric point pH, the snail mucin is positively charged and above, it is negatively charged. 3.5 Local anaestl~eticproperty It was shown that snail mucin had a local anaesthetic potential on the guinea pigs. Ihis is shown in Table 6. Maximum degree of anaesthesia was shown by the 320 mg / 2 ml sample of the stock solution. This effect may be due to the blockage of nerve impulse from the effector site. 3.6 'Toxicological studies 3.6.1 Acute toxicity test The extract appeared well tolerated, as the animals did not exhibit any symptoms of overt toxicity. The snail mucin is therefore non-toxic as no animal died. Table 6. Guinea pig wheal experiment Guinea pigs Snail-- much dose (mgI2mI) Degree of anaesthesia I 80 316 3.6.2 Chronic toxicity test 3.6.2.1 Haematoiogy The result of the packed cell volume (PCV) of the blood samples of the rat groups before and afier dosing with the snail mucin reveals an insignificant difference (p< 0.05) as shown in Fig. 10. The result of the erythrocyte sedimentation rate (ESR) also reveals an insignificant change (p < 0.05) in ESR values both before and after dosing with the snail mucin. The result is presentd in Fig1 1. ESR is an index of damage to vital organs or serious toxicity/infections. The lack of significant changes in ESR implies that the varied doses of the mucin extract caused very insignificant alterations in the vital organs - livers and kidneys of the rats (Fig. 11). The total leucocyte counts ('I'LC) per microlitre of blood shows that dosing the rats at 750 and 1500 mg/kg had insignificant alteration in the TLC but the 3000 mglkg dose had a significant fall in 7'1X (p < 0.05) of the rats. This fall is called leucopenia and is probably due to a fall in their absolute monocyte and absolute eosinophil counts (Fig. 12). There was thus no significant effect on the lymphocyte and neutrophil counts of all the rat groups, hence no effect on the immune system. The result of the absolute lymphocyte count (ALC) per microlitre of blood reveals also an insignificant difference (p < 0.05) in the lymphocyte counts of all the rats. This further attests to the fact that there was no effect on the immune system (Fig. 13). Mot~ocytesnre known to be involved in phagocytosis of large particles and eosinophils are usually involved in allergic reactions. The significant fall in absolute monocyte count (AMC) of the rats dosed with 3000 mg/kg of snail mucin (p < 0.01) could be attributed to the mobilization of these cells fbr the removal of the snail mucin from the body (Fig, 14). Fig. 15 shows the result of the absolute neutrophil counts (ANC) per microlitre of blood. This reveals an insignificant fall in neutrophils (p<0.05) in the rat groups as shown in the figure. 750mglkg bw 1500mglkg 3000mglkg None bw. bw. (Control) Graded doses of snail slime €3 Before dosing After dosa C ------ Fig 10. Packed cell volume (PCV) of albino rat groups given graded oral doses of snail mucin extract. 750mglkg bw 1500mglkg 3000mglkg None bw. bw. (Control) Graded doses of snail slime [I3 Before dosing After dosing] .. .-- -. .. - -. - -- .. -..- ...... -. -- .- .. . .- .- .. Iiig. 11. Erythrocyte sedimentation rate (ESR) of albino rat groups given graded oral doses of snail much extract. 750mglkg bw 1500mglkg bw. 3000mglkg bw. None (Control) Graded doses of snail sllme Eefore dosing After-- dosin4- - Fig. 12. Total leucocyte counts of albino rat groups given graded oral doses of snail much extract. $ ioooo ( 750mglkg bw 1500mglkg bw. 3000mglkg bw. None (Control) Graded doses of snail slime Fig. 13. Absolute lymphocyte cor~ntsof albino rat groups given graded oral doses of snail much extract. 750mglkg hw 1500mglkg bw. 3000mglkg bw. None (Control) Graded doses of snall slime -- LI3 Before Idosing O A-d Fig. 14. Absolute monocyte counts of albino rat groups given graded oral doses of snail much extract. 1500mglkg bw. 3000mglkg bw. None (Control) Graded doses of snail slime Fig. 15. Absolute neutroplli! counts of albino rat groups given graded oral doses of snail mucin extract. The eosinophils are involved in allergic response and stress conditions. The very high doses uscd for the study constituted a stress on the blood system of the rats such that there was significant fall (P < 0.01) ill the absolute eosinophil eounts (AEC) of all the rat groups that received the snail mucin. The fall tended to be dose-dependent. The extract either constituted a stress or may have induced an allergic response that led to the conlplete use up of eosinophils in groups B and C and a reasonably significant fall in the group A rats (Fig. 16). This fall contributed to the low counts obtained in the total leucocyte count of thc rat groups otherwise called leucopenia. The result is displayed below. -wnyu 3.6.2.2 Histopathology J"m The result of the histopathology is shown on the photomicrographs (Fig. 17). There were no significant histopathological changes on the kidney and liver of the rats that received the snail mucin. The hepatocytes and central veins of the livers of all the rats were intact and nornial only for a mild congestion of the central veins of the rats that received 3000 mglkg of snail mucin. This was probably a sign of increased blood supply needed to detoxify the body of the high dose (3,000 mglkg) of the mucin extract given to them. The kidneys of all the rat groups were normal. The tubular cells were intact and the glomeruli were normal. 3.7 Bioadhesive strength The result of bioadhesive strength of aqueous dispersion o f the snail much determine1 tensiomelry is shown in Fig. 18. The mucin had high bioadhesive strength attributed to the interaction between the functional group(s) in the snail mucin and the hydrophilic-OH and -NH2 groups of the mucus- gycoproteins resulting in the formation of strong bonds leading to high bond strengths. Invariably, it may also be due to the long contact time between the snail mucin and the mucus since increase in contact time favours bioadhesion provided excessive swelling leading to over-hydration does not occur [I 8,211. I 750mglkgLbw 1500mglkg bw. 3000mglkg bw. None (Conlrol) -100 1 Graded doses of snail slime Fig. 16. Absolute eositiophil counts of albino rat groups given graded oral doses of snail much extract. Normal liver Normal kidney Figure 17. Photomicrographs ofthe liver and the kidney. 1 2 3 4 5 Concentration (%wlv) Fig. 18. Effect of concentration on the bioadhesive strength of the snail mucin dispersions: 1 =4%wlv; 2=8%wlv; 3= 12%wlv; 4= 16%wlv; 5=20 %wlv 3.8 Evalr~ationof bioadhesive strength using coated glass beads Fig. 19 shows the result of the bioadhesive test using coated glass beads. Different solvents have varying effects on the percentage of glass beads detached from the layer. This gives an indication that different detaching solvents with different pH values have ideal regions in the mucosal cavity where drugs could be targeted. pH determines the solubility, stability and viscosity of a given material, and these affect the ease of absorption of drugs from the gastrointestinal tract into the blood system. The SGF used in this study has a pH of 1.2 while the SIF has a pH of 7.5. Figure 19 revealed high bioadhesive strength as shown by the 16 and 20 % w/v concentration of the mucin, which gave 100 % bioadhesion. The bioadhesive strength was very much higher when SGF was used as washing solvent implying that the snail mucin could be useful in formulating drugs that release primarily in the gastric mucosa. This is further supported by the observation made by Kellaway et al. [I 81 during a study of a 1 % gelatin gel at various pH values, where he concluded that a low pH favours bioadhesion. Invariably, it was inferred that the SIF with a pH of 7.5 f 0.1 weakened the bioadhesion of the snail mucin-coated glass beads by not offering much resistance to washing even at the highest concentration Fig. 20. At the highest coilcentration of 20 % wlv, SIF gave a bioadhesive value of 66.33 %, which is much less than the bioadhesion (86.67 %) offered by the 12 % w/v concentration of the mucin using SGF as the washing fluid. Therefore, it is meaningless to structure a dosage formulation with snail mucin to target entero (intestinal) release since tnucoadhesion at that site is not favoured. 3.9 Bioadhesive test on the granules The result of the bioadhesion so far supports the use of SGF as the detaching solvent (low pH). SGF was thus made use of here. Moreover, chlorpropamide used in the formulation is 2 3 4 Concentration (%wlv) Fig.19. Snail mucin concentration as affected by SGF washing 1 2 3 4 5 Concentration (%w/v) Fig. 20. Snail mucin concentration as affected by SIF washing shown to dissolve in dilute solutions of alkali hydroxides so that the SGF also favoured its use in the study. The result is presented in Fig. 2 1. The result of bioadhesion on the formulated granules indicated that granules formulated with snail mucin alone that is, batch 4 were more bioadhesive than those formulated with carbopol ultrez-10 polymer alone (batch 5) and thus had a higher percentagc of bioadhesion. Ilowever, there was optimum bioadhesion at equal combination of the Carbopol Ultrcz-I0 and snail mucin as in (batch 1). Maximum percentage bioadhesion was also achieved with Carbopol ultrez-I0 and snail mucin at a ratio of 2: 1 (batch 3), which gave exactly the same percentage of bioadhesion as in 0:l ratio (batch 4). Bioadhesive materials have been identified as being hydrophilic macromolecules containing numerous hydrogen bond-forming groups 1221. Snail mucin possesses hydrogen bond-forming groups hence favouring bioadhesion. Also, the bioadhesion of the granules unto the mucus membrane may be due to mechanical interlocking. These results show that chlorpropamide can be successfully delivered to the stomach by drug targeting, since bioadhesion, absorption and a possible prolonged effect on absorption can be achieved. 3.10 Granule micromeritics 'The properties of the formulated chlorpropamide granules are presented in Table 7. The range of granule flow rate was 1.43 - 2.50 glsec. The angle of repose, which indirectly quantifies powder flowability relates to inter-particle cohesion [3 11 and was in the range of 39.47 - 41.05'. This implies good flow properties. The bulk and tapped densities were within the ranges of 0.50 - 0.59 and 0.63 - 0.71 respectively. The Hausner's quotient was within the range of 1.20 - 1.26. An increase in consolidated bulk densities is advantageous in tableting [31]. For Hausner's quotient, values approximately 1.2 indicate good flowability. The percentage compressibility values of the granules lie between 16.90 and 20.63%. Materials that have values of 5 - 15, 12 - 16 and 18 - 21 % possibly have excellent, very good and fair flow behaviours respectively [3 11. Batch 1 Batch 2 Batch 3 Batch 4 Batch 5 Batches Fig. 21. Bioadhesive abilities of the Carbopol Ultrez 10IMucin granules [Batch 1 (1:1), Batch 2 (1:2), Batch 3 (2:1), Batch 4 (0:1), Batch 5 (1:0)] Table 7. Granule n~icromeritics ~/NO.Carbopol- Flow Angle of Bulk Tapped Hansner's Percentage niricin Rate Repose density density quotient compressibility ratio @Is) (degrees) (gw (ml) (HQ) (%) 'I'hose of values 23 - 35 O/o compressibility indicate poor flow. Therefore, the granules had excellent and good flow behaviours. 3.1 1 Effect an small intestinal motility The result of the charcoal meal transit is shown in Table 8. Motility of gastrointestinal smooth m~tsclesis affected from the stomach to the colon. Segmental contractions of the smooth muscle mix the intestinal contents. In general, the walls of the viscera are relaxed, and both tone and propulsive movements are diminished. Therefore, gastric emptying time is prolonged and intestinal transit time lengthened. Abnormal activity of the gut may cause accelerated, retarded or retrograde (reflux) movement of its contents. Blockade of muscarinic receptors has dramatic effects on motility and some of the excretory functions of the gut. The administration of snail mucin significantly (p < 0.05), reduced in a dose-related manner the charcoal meal transit although optimally at a dose of 400 mglkg. 'The inhibition thus produced was of 1 .I % whereas 10 mg of Atropine gave an inhibition of 1.0 % of the gut transport of the charcoal plug. This shows that snail mucin lowers intestinal motility at high doses. 3.12 Absolute drug content The active ingredient contents did not vary widely. The various batches had low standard deviations and thus conformed to Pharmacopoeia standard. The small content variations indicated good formulation, and may be as a result of minor weight variation. The results are shown in Table 9. 3.13 Release studies The release profile of chlorpropamide from the granules is shown in Fig. 22. It revealed a prolonged release of chlorpropamide in the granules. This is probably as a result of combined effect of increase in tortuosity on gelling [I241 and polymer - drug interaction (drug binding). There may have been iriteraction between chlorpropamide and carbopol-snail mucin copolymer Table 8. Effect of the snail mucin (extract) on small intestinal motility -. ------Dmg Dose (mglkg) Distance travelled (%) Tween 85 20 (mllkg) 46.2 * 3.0 Atropine 10 1 .O * 0.8 Extract 1 00 25.0 * 0.8 Extract 200 14.8 2.1 Extract 400 1.1 2~0.1 -- P < 0.05 Table 9. Absolute drug content of the granules Carbopol -. Batch Absorbance Absolute drug content snail n~ucin (mg) * S.D. comoosition 0 30 60 90 120 180 Time (min) Fig. 22. Release profile of chlopropamide from the granules in SIF (pH 7.5) containing Carbopol Ultrez-lOlsnail mucin combinations +Batch 1 (1:l) -0- Batch 2 (1:2) +Batch 3 (2:l) +Batch 4 (0:l) -8- Batch 5 (1:O) .- -.. --- - ... -- -- that induced slow release of chlorpropamide from the granules. 'The increase in tortuosity increases the path length available for drugs to diffuse out from the gel matrix. The granule batches retarded chlorpropamide release achieving maximum release of 98% after 180 mins. The release was fastest from the granules produced with snail mucin alone as evident from batch 4 (0: I), which gave a maxinlum percentage release of 98.00 O/u at 180 min. Batch 5 (1:O) granules which had Carbopol 1Jltrez-10 but no snail nlucin gave a much less percentage release of 58.17 at 180 min. Besides, the percentage release when Carbopol Ultrez-10 and snail mucin were in combinations of 1 : 1 (batch 1) and 2: 1 (batch 3) were reasonably high giving values of 71.06 'YO and 74.15 O/u respectively. All the granule batches retarded the release of chlorpropamide achieving a maximunl release of less than 100 %. The t5" for the release of chlorpropamide was achieved in 60 inin for granule batch 4 (0: I), 120 min for batches 1 (1 :I), batch 3 (2: I) and batch 5 (1:O). 'Ibis was not achieved for the granule batch 2 (1:2). A more useful comparative approach was tzo that is the time taken to release 20 % of chlorpropamide from the granules. The time taken to release 20 % of the drug was 15 min for batch 4,45 min for batches I, 3 and 5 then 60 niin for batch 2. These prolonged periods of release of active ingredient from the granules further attests to the purported use of these mucoadhesive materials (Carbopol Ultrez-I0 and snail mucin combinations) for the formulation of chlorpropamide as a prolonged release dosage form. The release result was further analysed using IIiguchi's square root model [121]. A plot of the amount of drug released against the square root of time when linear, indicates that diffusion is the predominant process of release. The entire granule batches showed linear plots except batch 2 granules (Fig. 23). This showed a biphasic linear lines signifying that at initial time intervals, the relcaw of drug was due to peripheral granules giving an initial straight line but difficulty in penetration of the SIF accounted for the break in linearity and the second linear segment was due to the release as a result of total penetration of the SIF. 'The release of chlorpropamide from the granule dosage form was also analysed using Fickian diflusion model to determine the mechanism of release of chlorpropamide from the Fig. 23. Higuchi's plot of the percentage of chlorpropamide released Batch 1 0 Batch 2 A Batch 3 A Batch 4 0 Batch 5 ------granules [122]. To understand the release mechanism of chlorpropamide from the granules, the release rate was described with the following equations: Eqn. 22 Eqn. 23 -Mf is the fraction of released drug at time r, K is n characteristic constant that incorporates the M stn~ctureand geometric characteristics of the release device and n is the release exponent indicative of the mechanism of release. As the K value becomes higher, the drug is released faster. The n value of 1 corresponds to zero-order release kinetics. 0.5 < rt < 1 means a non- Fickian (anomalous) release model and n = 0.5 indicates Fickian diffusion [122]. From the plot of M Log2 versus log t (Fig. 24), the kinetic parameters, 11 and k were calculated and presented in M Table 10. This shows that the n values of all the batches lay between 0.5 and 1 thus indicated that release of chlorpropamide from the bioadhesive granules followed the non-Fickian diffusion model (anon~alousbehaviour). However, n values for batches 1, 2, 3 and 5 were approaching unity and could be said to have exhibited almost zero-order kinetics. The K values for batch 4 was higher than others indicating faster release in that batch. This implies that chlorpropamide can suitably be administered orally with snail mucin as a binder - disintegrant. Fig. 24. Fick's plot of chlopropamide released from the granules n Batch 1 Batch 2 r Batch 3 A Batch 4 o Batch 5 Table 10. Release kinetic parameters Batch n K 2 CHAFTER FOUR SUMMARY AND CONCLUSIONS Snail nlucin forms dispersion in water. Its intended utilization in pharmaceutical preparation necessitated the study of the effects of various factors on the viscosity of the mucin. 'I'he flow properties of all liquid pharmaceutical preparations are of great importance in maintaining the urliformity and standard of these preparations. Perhaps, the most vivid example for the need of a thorough knowledge of flow characteristics is in the case of emulsions and suspensions in which retardation of separation of dispersed phase or increase in the viscosity of the dispersion medium is essential. However, snail mucin demonstrated a pseudoplastic type of flow probably due to the presence of long high molecular weight molecules in solution resulting in entanglement alongside the association of immobilized solvent that disentangles and aligns then1 in the direction of flow thus releasing the entrapped water upon the influence of shear. Increase in temperature decreases the viscosity of the aqueous snail muci~ldispersion due to rupture of sub-units held together by inter-chain disulphide bonds and some non-covalent interactions between the mucus glycoproteins resulting in loss of visco-elastic characteristic of the mucin. Electrolytes also reduce the viscosity of snail nlucin depending on the hydrolysis and precipitation of the mucin [A1203>CuS04>KC1]. This implies that snail mucin is anionic. Polymers increase the viscosity of snail mucin dispersion. Coarcervation was the process by which gelatin increased the viscosity. SCMC probably increased the molecular entanglement between the snail mncin and SCMC molecules. Carbopol-940 demonstrated greater entanglement leading to rlieological synergism. By implication, gelatin, SCMC and carbopol-940 can be enlployed as viscosity-enhancers in suspensions, tablet coating as binders or disintegrants in dosage formulations. The viscosity of aqueous snail mucin dispersion decreases on storage probably due to hydrolysis and decomposition by enzyme. Proteins are amphoteric polyelectrolytes and so carry both positive and negative charges. 'I'he charge on a protein is a factor of protein stability in solution, sine it prevents the agglomeration of protein particles and their precipitation. The isoelectric point (I.P.) of snail mucin is 3.4. At I.P., snail mucin dispersion was unstable, and easily deposited as a precipitate, especially in the presence of ethanol (a dehydrating agent). The molecular weight of snail mucin is 4,281 Daltons by gel permeation chromatography on cross-linked dextran G-200. The obvious effect a drug can have on a nerve is to block the condition of impulses along it. 'I'he twitch-response test in a guinea pig's skin assessed the duration and intensity of the effect of snail mucin although ignores differences in the rate of onset and inability to cross mucous surfaces. 'I'he maximum degree of anaesthesia was demonstrated by the 320 mg/2ml dose of snail mucin dispersion a~idtlie effect may be due to blockade of nerve impulse from the effector site. Snail ~nuciliis very safe and well tolerated in animals. Its effect on the hematological parameters evaluated in the rats indicated a fall in the total leucocyte count (Pc0.05) at a dose of 3000 mglkg. This fall is otherwise called leucopenia. Monocytes are involved in phagocytosis of large particles and eosinophils involved in allergic and stress conditions resulting in their significant fall (PcO.01) probably due to the mobilization of these cells for the removal of the high dose of the snail mucin from the body since the 3000 mglkg dose may have constituted a stress or may have induced an allergic response leading to a complete use of the eosinophils. However, there was no effect on the immune system. The effect of cumulative dosing of the snail mucin- tragacatli suspension on the organs of the liver and kidney histopathologically reveals insignificant change in tlie rat kidneys. The 3000 mg/kg dose, however, gave a mild congestian of the central veins of the liver although the liepatocytes were intact. This was probably a sign of increased blood supply needed to detoxify the body of the high mucin dose. 'The snail mucin had high bioadhesive strength of 1.80 - 4.5x10-~~m-' probably due to interaction of the mucus-mucus glycoproteins resulting in high bond strengths. It may also be due to the long contact time which favours bioadhesion provided over-hydration does not occur 1 181. The bioadhesion test on the coated glass beads reveled that the coated glass beads were more resistant to SGF washing (I6 and 20 % w/v each gave 100 % bioadhesion) than SIF. This implies that a low pH favours bioadhesion [18]. Moreover, snail mucin can be used as a binder to formulate a drug that primarily release in the stomach. The bioadhesion tests on chlorpropamide' granules show that snail mucin was more bioadhesive (94.1%) than Carbopol utrez-10 (80.5%). By implication, chlorproparnide can be successfiilly delivered to the stomach by drug targeting, since bioadhesion, absorption and a possible prolonged effect on absorption can be achieved. 'I'he chlorpropamide granules had good micromeritic properties [3 I]. Motility of the gastrointestinal smooth muscles is affected from the stomach to the colon of which segmental contractions mix the intestinal contents. When the walls of the visceral relax both tone and propulsive movements are diminished, thus gastric emptying time and intestinal transit time lengthen. However, abnom~algut activity may cause accelerated, retarded or retrograde (reflux) movement of its contents. Snail mucin, therefore, significantly (P<0.05) reduced in a dose-related manner the charcoal meal trranist implying that snail mucin may have caused blocked of n~uscarinicreceptors resulting in dramatic effects on motility and some of the excretory functions of the gut. The absolute drug content analysis of cldorpropamide granules conformed to acceptable standards. The release profile of the granules indicate a prolonged release of chlorpropamide probably as a result of combined effect of increase in tortuosity on gelling [124] and polymer-drug binding. The granule that had snail mucin alope gave higher drug release (98%) than Carbopol ultrez 10 (58.17%) over 180 inins. 'There may have been an interaction between the drug and the polymers that induced slow release of chlorpropamide from the granules. The increase in tortuosity increases the pathlength available for drugs to diffuse out From the matrix. By implication, snail mucin probably can be used as a disintegrant in the formulation of chlorpropamide granules. Further analysis of the chlorpropamide release was done using Higichi's square root mode [121]. This indicates that diffusion was the predominant process of release and the entire granule batches showed linear plots. Fickiqn diffusion model was also used b to determine the mechanism of release of chlorpropamide from the granules [122]. However, the release pattern followed the non-fickian model (anomalous behaviour) since the release exponent n, indicative of the mechanism lay between 0.5 and 1. However, since the n values of batches 1,2,3 and 5 were approaching unity, they could be said to have exhibited almost zero order kinetics. Since higher K values indicate faster release, then, batch 4 granules (only snail mucin) released faster. ?his implies that snail nii~cinacted as a binder-disintegrant in the chlorproaniide granules thus can be used alone to formulate chlorpropamide as well as other drugs provided such drugs primarily release in the stomach. Snail mucin forms dispersion in water. It is safe in animals. Polymers enhance its viscosity but age, electrolyte and temperature affect it. By implication, for suspensions to be prepared with snail mucin, thc drug has to be in the forn~of granules to be reconstituted immediately before use. Such suspension has to be stored in the refrigerator and used within 7 days. 'The molecular weight of snail mucin is 4,281 Daltons. Its isoelectric point is 3.4. This implies that at this pH, snail mucin has no net charge and is easily precipitated by dehydrating agents like ethanol, acetone, etc. These, however, should not be used as cosolvents in any preparation involving snail mucin. It has no serious hernatological or histopathological effect in rats. It has high bioadhesive strength attributed to interaction between functional groups as well as the long contact time. The mucosal pH affects solubility, stability and viscosity of a given material. These factors affect the ease of absorption of drugs from the Gl'T into the blood stream. However, low pH favours the bioadhesion of snail mucin indicating that drugs that primarily release in the gastric environment are possible candidates for formulation with snail mucin as an excipient in solid dosage form. The chlorpropamide granules had high bioadhesion (94.1 %) when formulated with snail mucin alone. The granules had good flow behaviours indicative of uniformity in contents and weight. There was small content variation indicative of good formulation. The granules containing snail mucin alone gave the highest release of 98 O/o over 180 min as compared to 58.17 % of Carbopol-ultrez 10 alone. This is to say that snail mucin perhaps may have acted 4s a release enhancer in the formulation. It is a local anaesthetic.achieving maximum aneasthesia at a dose of 160 mgtml dispersion. 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APPENDICES Beers plot of chlorpropamlde In SIF 0 1 2 3 4 5 6 Conc (nrg) Data for Beer's Plot ------Concentration (mg %) -1' Absorbance (229.7 nm) Slope, K = 0.1025X Data for the dissolution profile of chlorpropamide granules formidated with earbopol-ultrezl0 and snail much (D.F. = 10) Batch I ( I : 1) - Amount Percent In % Square root Time (Mins) released (mg released (%) (nm) unreleased .- -- %) 5 0.052 5.2 2.08 4.58 0.29 Batch 2 (1:2) .-- Amount Percent Time (Mins) Absorbance In % Square root released (mg released (%) (nm) unreleased %) 5 0.035 3.5 1.41 4.59 0.29 - 15 0.043 4.3 1.73 4.59 0.50 -- --7 30 - 0.250 25.0 10.04 4.50 0.71 45 0.4 10 41 .O 16.47 4.43 0.87 60 0.620 62.0 24.90 4.32 1 .OO ------.- - 90 0.690 69.0 27.71 4.28 1.22 ------120 0.750 75.0 30.12 4.25 1.51 150 0.760 76.0 30.53. 4.24 1.58 .- 180 0.770 77.0 30.92 4.24 1.73 Batch 3 (2: 1) Percent Time (Mins) In % Square root r-- unreleascd Batch 4 (0:I) -~--- Time (Mins) Absorbance r1-Treleased (mg released (%) unreleased In % 1;"are root (nm) Batch 5 (1 :0) Amount Percent In % Square root unreleased Data for 17ick's Plot Log --M, M I Batch 1 Effect of temperature on the viscosity of 20 % wlv dispersion of snail mucin. Temp. ("C) Average instrument reading Viscosity (mPas) --. --- 32 18.0 304.43 Effect of electrolytcs on the viscosity of 20 % w/v dispersion of snail mucin 1 I Instrument readings Viscosities (mPas) I Without KCI --electrobe CuSOl AI2O3 )us snail mucin dispersions. Instr~~nientreading of the snail mucin I Viscosities (mPas) conc. ("/o w/v) I Gel permcation chromatography on Sephadex (3-200 Log Mol. -darns V (ml) Mol. Wt. Kav 7 I Wt. 1 Blue dextran Methyl rctl 1 :: 1 2,1W:.ry 1 2.4730 Dovine Scrum Albumin 34 64,000 1.026 . (BsA) I Ribonucleasc 1 39.4 1 13,700 1 1.351 1 Ovalbumin 1 35.5 1 45.000 11.116 I Snail mttcin 1 44 1 4,281 .I4 1 1.629 Packed Cell Volume (PCV) %, + S.D. 1 Drug Before dosing After dosing Extract r91 49-50 * 2.07 1 Extract 48,33 :k 1.75 48.33 * 2.04 Extract 'Tragacanth placebo 48.33 -1: 2.58 49.17 * 2.64 -- P < 0.05 [E:xtract herc represents snail mucin] Erythrocyte Sedimentation Rate (ESR) mni; * S.D. Drug Before dosing AAcr dosing - Extract 1.07 * 0.08 1.07 + 0.33 -. . Extract 1.08 i 0.13 1.05 0.22 Extract 1.17* 0.24 1.05 * 0.12 Tragacanth placcbo 1.05 * 0.3 1 l.lO* 0.18 P < 0.05 Total Lcucocyte Counts per microlitre of blood; * S.D. Drug Before dosing ARer dosing Extract 1 16,866.67i 3,276.69 / Extract I 15,925.00* 3,5 10.09 I 14,791.67 + 1,957.40 Tragacanth placebo 15,491.67i 2,008.83 14,94 1.67 + 1,275.70 ab: P .: 0.05 Absolute Lymphocyte Counts per microlitre of blood; + S.D. Before dosing After dosing I Extract I 10,049.50 1 155.43 I 10,012.75 1932.58 LA-Tragacanth placebo 10,266.58 * 655.32 10,941.83 + 1403.01 Absolute Monocyte Counts pcrmicrolitre,of blood rt S.D. ---.- -- Drug 7-Before dosing Afier dosing Extract 2815.83 :k 1406.68 2083.67 + 738.00 2123.33 rt 891 -53 1501.67 f 447.19 -- Extract 2 1 1 2.7Sa f 493.5 1 1454.92~+ 214.13 Tragacanth placebo 2525.42 * 1036.01 1902.67 f 425.42 .1 ab : p<0.01. p:0.05 Absolute Ncutrophil Counts per niicrolitre of blood k S.D. I Drug Bcfore dosing Afier dosing Extract ----- Tragacanth placcbo 2565.50 rt 10 13.55 2 1 16.42 f 3 12.97 P < 0.05 Absolutc Eosinophil Counts per niicrolitrc of blood * S.D. - Before dosing Afier dosing Extract 1 429.58' * 366.18 1 24.25; 59.40 Extract 406.1 7a k 27 71.30 Extract 398.25' A 229.28 oh Tragacanth placebo 393.38' * 198.61 31 1.17af 27.87 ab : P < 0.01 Tensiometric determination of bioadhesivc strength Conc. C)f snail fiGge interfacial- I Standard deviation ( Calculated Tension I nirrciti (% wlv) tension (degrees) ( X ') (S.D.) I 0')(~m-') -- I I Effect of concentration of snail mucin (% wlv) on the bioadhesion of coated glass beads using siniulated gastric fluid (SGF) as the detaching solvent. - -- Conc. of ~nail--(& beads Var, of Standard error Percentage Mucin (% wlv) ,,,.-I of the mean bioadhesion (%) undetached (X") I (SEM) I I Effect of concentration of snail mucin (% wlv) on the bioadhesion of coated glass beads using simulated intestinal fluid (SIF) as the detaching solvent. --A- / Conc. of Snail 1 Mean beads 1 Var. of I Standard error I Percentage mean of the mean bioadhesionV(%) I (SEW I Rioadhesion ability of the formulated granules using SGF as the detaching solvent. Polymer-Drug Weight of Weight of Percentage of Batches ratio detached granules undetached granule .-----. ( g granules (g) undetached (%) 1 1:l 0.08 15 0.9 185 91.9 Tlic relationship between speed and rate of shear using a 20 % w/v dispersion of mucin -- Speeds instrument reading Viscosity (mPas) 1 Typc of flow exhibitcd by a fresh 2 % w/v dispersion of snail mucin Shcnring stress Rate of shear (mPas) 1 Effect of concentration on the viscosity of thc snail mucin dispersions ---. Viscosities (% Specds (mPas) of the various snail mucin conc w/v) 2 4 8 12 I6 20 I 3.38 8.46 16.91 18.60 22.00 25.37 2 32.13 33.83 35.52 42.28 47.36 50.74 3 48.20 50.74 60.89 67.65 76.12 82.03 ------5 50.74 67.65 76.12 101.48 118.39 138.68 --- -. .------9 84.56 109.93 152.22 180.46 202.93 248.1 I Effect of polymcrs on the viscosity of 20% w/v dispersion of snail mucin - -- -7-instrument readings Viscosities-7 (mPas) polymer Turbidity of snail mucin dispcrsion PH Without ethanol addition (ml) With ethanol additlon (ml)